Dmrt1 is the only male pathway gene tested indispensable for sex determination and functional testis development in tilapia.

Sex is determined by multiple factors derived from somatic and germ cells in vertebrates. We have identified amhy, dmrt1, gsdf as male and foxl2, foxl3, cyp19a1a as female sex determination pathway genes in Nile tilapia. However, the relationship among these genes is largely unclear. Here, we found that the gonads of dmrt1;cyp19a1a double mutants developed as ovaries or underdeveloped testes with no germ cells irrespective of their genetic sex. In addition, the gonads of dmrt1;cyp19a1a;cyp19a1b triple mutants still developed as ovaries. The gonads of foxl3;cyp19a1a double mutants developed as testes, while the gonads of dmrt1;cyp19a1a;foxl3 triple mutants eventually developed as ovaries. In contrast, the gonads of amhy;cyp19a1a, gsdf;cyp19a1a, amhy;foxl2, gsdf;foxl2 double and amhy;cyp19a1a;cyp19a1b, gsdf;cyp19a1a;cyp19a1b triple mutants developed as testes with spermatogenesis via up-regulation of dmrt1 in both somatic and germ cells. The gonads of amhy;foxl3 and gsdf;foxl3 double mutants developed as ovaries but with germ cells in spermatogenesis due to up-regulation of dmrt1. Taking the respective ovary and underdeveloped testis of dmrt1;foxl3 and dmrt1;foxl2 double mutants reported previously into consideration, we demonstrated that once dmrt1 mutated, the gonad could not be rescued to functional testis by mutating any female pathway gene. The sex reversal caused by mutation of male pathway genes other than dmrt1, including its upstream amhy and downstream gsdf, could be rescued by mutating female pathway gene. Overall, our data suggested that dmrt1 is the only male pathway gene tested indispensable for sex determination and functional testis development in tilapia.

CCND1 copy number increase and cyclin D1 expression in acral melanoma: a comparative study of fluorescence in situ hybridization and immunohistochemistry in a Chinese cohort.

BACKGROUND: CCND1 copy number increase is characteristic of acral melanoma and is useful in distinguishing benign and malignant acral melanocytic lesions. Increase of the gene copy number may result in protein overexpression. This raises the possibility that detection of high expression of cyclin D1 by immunohistochemistry (IHC) may be used as a surrogate for direct evaluation of increase in the CCND1 gene copy number. METHODS: We examined increases in CCND1 copy number with fluorescence in situ hybridization (FISH), and examined cyclin D1 protein expression with IHC in 61 acral melanomas. RESULTS: Using FISH, 29 acral melanomas (29/61, 47.5%) showed increase in the CCND1 copy number, including 8 (8/61, 13.1%) which showed low-level increase in the CCND1 copy number and 21 (21/61, 34.4%) with high-level increase in the CCND1 copy number. By analysis of IHC, the median IHC score was 15% (range: 1-80%) in acral melanomas with no CCND1 copy number alteration. In acral melanomas with low-level CCND1 copy number increase, the median IHC score was 25% (range: 3-90%). In acral melanomas with high-level CCND1 copy number increase, the median IHC score was 60% (range: 1-95%). Comparing FISH and IHC, cyclin D1 protein expression level has no corelation with the CCND1 copy number in acral melanomas which have no CCND1 copy number alteration and low-level CCND1 copy number increase (P = 0.108). Cyclin D1 protein expression level correlated positively with CCND1 copy number in acral melanomas with high-level CCND1 copy number increase (P = 0.038). The sensitivity, specificity and positive predictive value of using cyclin D1 IHC to predict CCND1 FISH result was 72.4, 62.5 and 63.6%. Increase in CCND1 copy number was associated with Breslow thickness in invasive acral melanoma. CONCLUSION: High-level increase in the CCND1 copy number can induce high cyclin D1 protein expression in acral melanomas. However low-level increase and normal CCND1 copy number have no obvious correlation with protein expression. Cyclin D1 IHC cannot serve as a surrogate for CCND1 FISH in acral melanomas.

Germline sexual fate is determined by the antagonistic action of dmrt1 and foxl3/foxl2 in tilapia.

Germline sexual fate has long been believed to be determined by the somatic environment, but this idea is challenged by recent studies of foxl3 mutants in medaka. Here, we demonstrate that the sexual fate of tilapia germline is determined by the antagonistic interaction of dmrt1 and foxl3, which are transcriptionally repressed in male and female germ cells, respectively. Loss of dmrt1 rescued the germ cell sex reversal in foxl3Δ7/Δ7 XX fish, and loss of foxl3 partially rescued germ cell sex reversal but not somatic cell fate in dmrt1Δ5/Δ5 XY fish. Interestingly, germ cells lost sexual plasticity in dmrt1Δ5/Δ5 XY and foxl3Δ7/Δ7 XX single mutants, as aromatase inhibitor (AI) and estrogen treatment failed to rescue the respective phenotypes. However, recovery of germ cell sexual plasticity was observed in dmrt1/foxl3 double mutants. Importantly, mutation of somatic cell-specific foxl2 resulted in testicular development in foxl3Δ7/Δ7 or dmrt1Δ5/Δ5 mutants. Our findings demonstrate that sexual plasticity of germ cells relies on the presence of both dmrt1 and foxl3. The existence of dmrt1 and foxl3 allows environmental factors to influence the sex fate decision in vertebrates.

Retracted: Allopregnanolone restores the tyrosine hydroxylase-positive neurons and motor performance in a 6-OHDA-injected mouse model.

AIMS: It has been reported that allopregnanolone (APα) promotes the neurogenesis of the neural progenitor cells (NPCs) in the subventricular zone (SVZ) and prevents the decrease of dopaminergic neurons in 6-hydroxydopamine (6-OHDA)-treated mice by binding to γ-aminobutyric acid A receptor (GABAAR) and then opening voltage-gated L-type Ca2+ channel, but the underlying mechanisms remain elusive. The aim of this study was to explore the possible involvement of GABAAR and calcium/calmodulin-dependent protein kinase II delta 3 (CaMKIIδ3) in this process. METHODS: 6-OHDA-treated mice and primary cultured midbrain cells were administrated with APα and GABAAR antagonist bicuculline (Bic), and the proliferation and differentiation of NPCs, the tyrosine hydroxylase (TH)-positive neurons and their fibers, the expression levels of CaMKIIδ3 and brain-derived neurotrophic factor (BDNF), and motor functions were measured using ELISA, immunohistochemical staining, real-time RT-PCR, Western blot, and behavioral test. RESULTS: Allopregnanolone significantly promoted the phosphorylation of cytoplasmic CaMKIIδ3 and its nuclear translocation by binding to GABAAR, which, in turn, increased the expression levels of BDNF. This may account for the findings that the exogenous APα enhanced the proliferation and differentiation of NPCs, and ameliorated the nigrostriatal system and behavioral performance in 6-OHDA-treated mice. CONCLUSIONS: Allopregnanolone may directly activate GABAAR, which, in turn, enhance the proliferation and differentiation of NPCs via upregulating the expression levels of CaMKIIδ3, and finally contribute to the restoration of dopaminergic neurons in 6-OHDA-treated mice.

Regulation of spermatogenesis and reproductive capacity by Igf3 in tilapia.

A novel insulin-like growth factor (igf3), which is exclusively expressed in the gonads, has been widely identified in fish species. Recent studies have indicated that Igf3 regulates spermatogonia proliferation and differentiation in zebrafish; however, detailed information on the role of this Igf needs further in vivo investigation. Here, using Nile tilapia (Oreochromis niloticus) as an animal model, we report that igf3 is required for spermatogenesis and reproduction. Knockout of igf3 by CRISPR/Cas9 severely inhibited spermatogonial proliferation and differentiation at 90 days after hatching, the time critical for meiosis initiation, and resulted in less spermatocytes in the mutants. Although spermatogenesis continued to occur later, more spermatocytes and less spermatids were observed in the igf3-/- testes when compared with wild type of testes at adults, indicating that Igf3 regulates spermatocyte to spermatid transition. Importantly, a significantly increased occurrence of apoptosis in spermatids was observed after loss of Igf3. Therefore, igf3-/- males were subfertile with drastically reduced semen volume and sperm count. Conversely, the overexpression of Igf3 in XY tilapia enhanced spermatogenesis leading to more spermatids and sperm count. Transcriptomic analysis revealed that the absence of Igf3 resulted in dysregulation of many genes involved in cell cycle, meiosis and pluripotency regulators that are critical for spermatogenesis. In addition, in vitro gonadal culture with 17α-methyltetosterone (MT) and 11-ketotestosterone (11-KT) administration and in vivo knockout of cyp11c1 demonstrated that igf3 expression is regulated by androgens, suggesting that Igf3 acts downstream of androgens in fish spermatogenesis. Notably, the igf3 knockout did not affect body growth, indicating that this Igf specifically functions in reproduction. Taken together, our data provide genetic evidence for fish igf3 in the regulation of reproductive capacity by controlling spermatogenesis.

[Herbal-cake-partitioned moxibustion of "Shenque" (CV8) has a relative specific effect in relieving abdominal pain and in regulating neuroendocrine-immune network in primary dysmenorrhea rats].

OBJECTIVE: To observe the effect of herbal-cake-partitioned moxibustion (HCPM) of "Shenque" (CV8) and "Daheng" (SP15) on abdominal pain, plasma ß-endorphin (ß-EP), uterine prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α) levels, as well as splenetic natural killer cell (NK cell) activity in primary dysmenorrhea (PD) rats, so as to explore the specificity of acupoint function and the underlying mechanisms of moxibustion in relieving dysmenorrhea. METHODS: A total of 40 female rats were randomized into blank control, model, CV8-direct moxibustion, CV8-HCPM and SP15-HCPM groups (n＝8 rats in each). The PD model was established by subcutaneous injection of estradiol benzoate injection (0.2-0.5 mg/rat) for 10 consecutive days and intraperitoneal injection of oxytocin (2 U) 24 h after the last subcutaneous injection. Moxibustion or herbal-cake (composed of Radix Angelicae Sinensis, Rhizoma Chuanxiong, Radix Paeoniae Rubra, Cortex Cinnamomi, etc.)-partitioned moxibustion was applied to CV8, SP15 or umbilicus respectively for 7 moxa-cones every time, once daily for 10 successive days. The rats of the control and model groups were also restrained as those in the moxibustion groups. The writhing times within 30 minutes was recorded and the contents of plasma ß-EP, uterine PGE2 and PGF2α were detected by ELISA, and NK cell activity was detected using MTT. RESULTS: Compared with the control group, the writhing times and the content of PGF2α in the uterus tissue were significantly increased in the model group (P<0.01), while the contents of plasma ß-EP, uterine PGE2 and splenetic NK cell activity were significantly decreased (P<0.01). In comparison with the model group, the writhing times and uterine PGF2α content were obviously down-regulated in the SP15-HCPM, CV8-direct moxibustion and CV8-HCPM groups (P<0.05, P<0.01), and the contents of plasma ß-EP and uterine PGE2, and splenetic NK cell activity were significantly increased (P<0.05, P<0.01). The therapeutic effects of CV8-HCPM group were significantly superior to those of SP15-HCPM and CV8-direct moxibustion groups in lowering writhing times and PGF2α level, and in up-regulating ß-EP, PGE2 (P<0.05, P<0.01). The NK cell activity of CV8-HCPM group was significantly increased compared with that in the SP15-HCPM group(P<0.05). No significant differences were found between the SP15-HCPM and CV8-direct moxibustion groups in the levels of writhing times, plasma ß-EP and uterine PGE2, PGF2α contents and splenetic NK cell activity (P>0.05). CONCLUSION: Moxibustion of both CV8 and SP15 can relieve abdominal pain in PD rats, which may be closely associated with its effect in suppressing PD-induced decrease of plasma ß-EP and uterine PGE2 levels and splenetic NK cell activity and increase of uterine PGF2α. The therapeutic effect of CV8-HCPM is obviously better than that of SP15-HCPM and CV8-direct moxibustion.

Allopregnanolone Modulates GABAAR-Dependent CaMKIIδ3 and BDNF to Protect SH-SY5Y Cells Against 6-OHDA-Induced Damage.

Allopregnanolone (APα), as a functional neurosteroid, exhibits the neuroprotective effect on neurodegenerative diseases such as Parkinson's disease (PD) through γ-aminobutyric acid A receptor (GABAAR), but it has not been completely understood about its molecular mechanisms. In order to investigate the neuroprotective effect of APα, as well as to clarify its possible molecular mechanisms, SH-SY5Y neuronal cell lines were incubated with 6-hydroxydopamine (6-OHDA), which has been widely used as an in vitro model for PD, along with APα alone or in combination with GABAAR antagonist (bicuculline, Bic), intracellular Ca2+ chelator (EGTA) and voltage-gated L-type Ca2+ channel blocker (Nifedipine). The viability, proliferation, and differentiation of SH-SY5Y cells, the expression levels of calmodulin (CaM), Ca2+/calmodulin-dependent protein kinase II δ3 (CaMKIIδ3), cyclin-dependent kinase-1 (CDK1) and brain-derived neurotrophic factor (BDNF), as well as the interaction between CaMKIIδ3 and CDK1 or BDNF, were detected by morphological and molecular biological methodology. Our results found that the cell viability and the number of tyrosine hydroxylase (TH), bromodeoxyuridine (BrdU) and TH/BrdU-positive cells in 6-OHDA-treated SH-SY5Y cells were significantly decreased with the concomitant reduction in the expression levels of aforementioned proteins, which were ameliorated following APα administration. In addition, Bic could further increase the number of TH or BrdU-positive cells as well as the expression levels of aforementioned proteins except for TH/BrdU-double positive cells, while EGTA and Nifedipine could attenuate the expression levels of CaM, CaMKIIδ3 and BDNF. Moreover, there existed a direct interaction between CaMKIIδ3 and CDK1 or BDNF. As a result, APα-induced an increase in the number of TH-positive SH-SY5Y cells might be mediated through GABAAR via Ca2+/CaM/CaMKIIδ3/BDNF (CDK1) signaling pathway, which would ultimately facilitate to elucidate PD pathogenesis and hold a promise as an alternative therapeutic target for PD.

Fluorescence in situ hybridisation as an ancillary tool in the diagnosis of acral melanoma: a review of 44 cases.

Acral melanoma is associated with outcomes which are more unfavourable than those of other melanoma subtypes, and acral melanoma has higher mortality. However, histological distinction of acral melanoma from acral naevi may be difficult. Fluorescence in situ hybridisation (FISH) targeting specific genes has been used as an ancillary method for differential diagnosis of melanocytic tumours, but most previous studies have focused on non-acral lesions which may have genetic alterations different from acral lesions. We evaluated use of multi-site FISH in the diagnosis of acral melanoma in a series of 82 acral melanocytic tumours. Two probe groups were applied. Probe set 1 involved a 4-probe FISH targeting 6p25 (RREB1), CEP6 (centromere 6), 6q23 (MYB) and 11q13 (CCND1). Probe set 2 involved a 3-probe FISH targeting 8q24 (MYC), 9p21 (CDKN2A) and CEP9 (centromere 9). In 44 primary acral melanomas, sensitivity was 70.5% (31/44) using probe set 1 alone, and 59.1% (26/44) using probe set 2 alone. When both probe sets were combined, sensitivity increased to 88.6% (39/44). The frequency of each gene alteration was as follows: MYC gain in 54.5% cases (24/44), RREB1 gain in 52.3% cases (23/44), CCND1 gain in 45.4% cases (20/44), MYB loss relative to CEP6 in 25.0% cases (11/44), and CDKN2A homozygous deletion in 20.5% cases (9/44). For lesions with both in situ and invasive disease, FISH findings in these two components were similar. No gene alterations were detected in any of 36 benign acral naevi. In this study FISH exhibited sensitivity and specificity in diagnosis of acral melanoma which allows its application as an auxiliary diagnostic test in acral melanocytic tumours.

[Effect of curcumin on the learning, memory and hippocampal Ca+/CaMK II level in senescence-accelerated mice].

OBJECTIVE: To explore effect of curcumin in different concentrations on learning and memory of senescence-accelerated mice (SAM) and their possible mechanisms. METHODS: Mice were randomly divided into six groups: the SAMR1 normal control group, the SAMP8 model control group, the SAMP8 + solvent (the peanut oil) control group, SAMP8 + low, middle and high dose curcumin groups. Mice were gastrogavage for 25 successive days. On the next day of ending the experiment, changes of learning and memory in mice of each group were observed by Morris water maze. The hippocampal [Ca2+] was determined. Expressions of hippocampal calmodulin-dependent protein kinase II (CaMK II) and Calmodulin (CaM) mRNA were detected using Western blot and reverse transcription polymerase chain reaction (RT-PCR) respectively. RESULTS: The latency to find the hidden platform was remarkably prolonged, the hippocampal [Ca2+]i was markedly increased, the expression of CaMK II in the hippocampal membrane and the level of hippocampal CaM mRNA were significantly reduced in the SAMP8-model control group (P < 0.05, P < 0.01). The latency to find the hidden platform was remarkably shortened in the SAMP8 + middle dose curcumin and the SAMP8 + high dose curcumin groups (P < 0.01). The hippocampal [Ca2+]i was markedly lowered, the expression of CaMK II in the hippocampal membrane and the level of hippocampal CaM mRNA obviously increased in the SAMP8 + low, middle and high dose curcumin groups (P < 0.05, P < 0.01). CONCLUSION: Curcumin could improve learning and memory Ca2+/capacities of SAM by lowering hippocampal [Ca2+] overload, increase the hippocampal CaM mRNA level and CaMK II expression in the hippocampal dose-dependently.

[Effect of Rhizoma curcumae oil on the learning and memory in rats exposed to chronic hypoxia and the possible mechanisms.].

The effect of Rhizoma curcumae oil on the learning and memory in rats exposed to chronic hypoxia and the possible mechanisms were investigated. The rats were divided randomly into 5 groups (14 animals in each group): control, chronic hypoxia, chronic hypoxia with low (5 mg/kg body weight), middle (10 mg/kg body weight) and high (20 mg/kg body weight) concentrations of Rhizoma curcumae oil injection. The animals undergoing chronic hypoxia were exposed to hypoxia in a hypoxic chamber containing 10% O(2) and 5% CO(2) for 10 h/d, lasting 28 d. Morris water maze (MWM) test was used to obtain the scores of leaning and memory. The superoxide dismutase (SOD) activity and malonaldehyde (MDA) content were determined in the serum and hippocampus as well as [Ca(2+)](i) in the hippocampus. The expression of phosphorylated Ca(2+)/calmodulin-dependent protein kinase II (p-CaMKII) in the hippocampus was evaluated by using immunohistochemistry and Western blot. Compared with the control group, the chronic hypoxia group showed the following changes: (1) The escape latency to the hidden platform was remarkably prolonged (P<0.05); (2) The content of MDA and [Ca(2+)](i) were obviously higher, but the activity of SOD and the expression of p-CaMKII were significantly lower (P<0.05, P<0.01). Compared with the chronic hypoxia group, groups with Rhizoma curcumae oil injection had the following changes: (1) The escape latency to the hidden platform was remarkably shorter in 10, 20 mg/kg body weight groups (P<0.05); (2) The content of MDA and [Ca(2+)](i) were markedly decreased in 5, 10, 20 mg/kg body weight groups (P<0.05, P<0.01), but the activity of SOD in the serum and the expression of p-CaMKII were significantly higher in 10, 20 mg/kg body weight groups (P<0.05, P<0.01). The results showed that the capacity of learning and memory was degraded following chronic hypoxia. The decrease in MDA content and [Ca(2+)](i) and (or) the increase in SOD activity and p-CaMKII expression might participate in the enhancing effect on learning and memory induced by Rhizoma curcumae oil.

Changes of learning, memory and levels of CaMKII, CaM mRNA, CREB mRNA in the hippocampus of chronic multiple-stressed rats.

BACKGROUND: The effect of chronic stress on cognitive functions has been one of the hot topic in neuroscience. But there has been much controversy over its mechanism. Such single stressor applied in the past could not simulate complicated living circumstances that people confronted with. The aim of this study was to investigate the effects of chronic multiple-stress on learning and memory as well as on the levels of calcium/calmodulin-dependent protein kinase II (CaMKII), calmodulin (CaM) mRNA, and cAMP-response element binding protein (CREB) mRNA in the hippocampus of rats. METHODS: The rats were divided randomly into stressed and control groups. The stressed group was given chronic multiple-stress for 6 weeks to set up a chronic multiple-stressed model. The rats' performance of spatial learning and memory was tested using Morris Water Maze (MWM) and Y-maze. Meanwhile, the expressions of CaMKII, CaM mRNA and CREB mRNA of rats' hippocampus were detected by immunohistochemistry, Western blot and reverse transcription-polymerase chain reaction (RT-PCR), respectively. In addition, the width of synaptic cleft and the thickness of post-synaptic densities (PSD) were observed in the hippocampal CA3 region of rats by electron microscopy. RESULTS: After exposure to chronic multiple-stress for 6 weeks, the ability of learning and memory of the stressed group was higher than that of the control group (P < 0.05, P < 0.01). The width of synaptic cleft was smaller and the thickness of PSD was larger in the hippocampal CA3 region of the stressed group than in that of the control group (P < 0.01). The CaMK II immunostaining of the stressed group was stronger than that of the control group in the stratum radiatum and oriens of the hippocampal CA1 and CA3, especially in the stratum oriens. Quantitative analysis indicated that the expression of CaMK II, CaM mRNA, and CREB mRNA in the hippocampus of the stressed group was higher than that of the control group (P < 0.05, P < 0.01). CONCLUSIONS: The capacity of learning and memory can be enhanced after chronic multiple-stress. The increased levels of CaMK II, CaM mRNA, and CREB mRNA may contribute to the enhancing effect of chronic multiple-stress on learning and memory.