The double sex and mab-3 related (DM) transcription factor 1 (dmrt1) plays an important role in testicular differentiation. Here, we report cloning of multiple dmrt1s, a full-length and two alternative spliced forms from adult catfish (Clarias gariepinus) testis, which encode predicted proteins of 287 (dmrt1a), 253 (dmrt1b) and 233 (dmrt1c) amino acid residues respectively. Interestingly, dmrt1c lacks the majority of the DM domain. Multiple dmrt1s (dmrt1a and dmrt1c) were obtained from Clarias batrachus as well. Tissue distribution (transcript and protein) of catfish dmrt1 revealed exclusive expression in testis. Semi-quantitative RT-PCR revealed the presence of multiple dmrt1s with high levels of dmrt1a in adult testis but not in ovary. Real-time RT-PCR analysis during testicular cycle showed higher levels of dmrt1 transcripts in preparatory and pre-spawning when compared with spawning and post-spawning phases. Immunocytochemical and immunofluorescence localization revealed the presence of catfish Dmrt1 protein in spermatogonia and spermatocytes, which indicates plausible role in spermatogenesis. Histological analysis indicated initiation of gonadal sex differentiation in catfish around 40-50 days after hatching. The potential role for dmrt1 in testicular differentiation is evident from its stage-dependent elevated expression in developing testis. Furthermore, dimorphic expressions of dmrt1s were evident at different stages of gonadal development or recrudescence in catfish. Treatment of methyl testosterone (MT) during early stages of gonadal sex differentiation resulted in adult males. Interestingly, we also obtained MT-treated fishes having ova-testis gonads. Analysis of dmrt1, sox9a, foxl2 and cyp19a1 expression patterns in MT-treated gonads revealed tissue-specific pattern. These results together suggest that multiple dmrt1s are testis-specific markers in catfish.

Gonadotropin-regulated testicular RNA helicase (GRTH/DDX25) is a testis specific member of the DEAD-box family of RNA helicases expressed in meiotic and haploid germ cells which plays an essential role in spermatogenesis. There are two species of GRTH the 56 kDa non-phospho and 61 kDa phospho forms. Our early studies revealed a missense mutation (R242H) of GRTH in azoospermic men that when expressed in COS1-cells lack the phospho-form of GRTH. To investigate the role of the phospho-GRTH species in spermatogenesis, we generated a GRTH knock-in (KI) transgenic mice with the R242H mutation. GRTH-KI mice are sterile with reduced testis size, lack sperm with spermatogenic arrest at round spermatid stage and loss of the cytoplasmic phospho-GRTH species. Electron microscopy studies revealed reduction in the size of chromatoid bodies (CB) of round spermatids (RS) and germ cell apoptosis. We observed absence of phospho-GRTH in the CB of RS. Complete loss of chromatin remodeling and related proteins such as TP2, PRM2, TSSK6 and marked reduction of their respective mRNAs and half-lives were observed in GRTH-KI mice. We showed that phospho-GRTH has a role in TP2 translation and revealed its occurrence in a 3′ UTR dependent manner. These findings demonstrate the relevance of phospho-GRTH in the structure of the chromatoid body, spermatid development and completion of spermatogenesis and provide an avenue for the development of a male contraceptive.

Prolactin (PRL) serves a critical role in breast cancer progression via activation of its cognate receptor. These studies reveal up-regulation of PRLR gene expression by PRL in absence of estradiol in MCF-7 and T47D breast cancer cells. PRL/PRLR via activation of STAT5 that binds a GAS-element in the PRLR gene and the participation of ERα stimulates PRLR transcription/expression. PRL/PRLR induces phosphorylation of ERα through the JAK2/PI3K/MAPK/ERK and JAK2/HER2 activated pathways. The increased recruitment of phospho-ERα, induced by PRL to Sp1 and C/EBPβ at PRLR promoter sites, is essential for PRL-induced PRLR transcription. This recruitment is prevented by blockade of PRL expression using RNA interference or ERα phosphorylation with specific inhibitors of PI3K and ERK. Direct evidence is provided for local actions of PRL, independent of estradiol, in the up-regulation of PRLR transcription/expression by an activation-loop between STAT5 and the phospho-ERα/Sp1/C/EBPβ complex with requisite participation of signaling mechanisms. PRL's central role in the up-regulation of PRLR maximizes the action of the endogenous hormone. This study offers mechanistically rational basis for invasiveness fueled by prolactin in refractory states to adjuvant therapies in breast cancer.

No abstract

In the present study the expression of 13 genes known to be involved in sex differentiation and steroidogenesis in catfish was analyzed during gonadal ontogeny by quantitative real-time RT-PCR. Dmrt1 and sox9a showed exclusive expression in male gonads while ovarian aromatase (cyp19a1) and foxl2 were abundant in differentiating female gonads. Most of the genes related to steroidogenesis were expressed only after gonadal differentiation. However, genes coding for 3β-hydroxysteroid dehydrogenase (3β-hsd), 17α-hydroxylase/C17–20 lyase type 1 (cyp17) and steroidogenic acute regulatory protein (star) were barely detectable during gonadal differentiation. Ovarian aromatase, cyp19a1, which is responsible for estradiol-17β biosynthesis in females, was expressed very early in the undifferentiated gonads of catfish, around 30–40 days post hatch (dph). The steroidogenic enzyme, 11β-hydroxylase (cyp11b1) required for the production of 11-ketotestosterone (11-KT) was expressed only after differentiation of testis. These results suggest that estradiol-17β has a critical role in ovarian differentiation, while the role of 11-KT in testicular differentiation is doubtful. In conclusion, dimorphic expression of dmrt1 and sox9a in gonads during early development is required for testicular differentiation, and sex-specific expression of cyp19a1 and foxl2 in females plays a critical role in ovarian development. Our study reveals that the critical period of gonadal differentiation in catfish starts around 30–40 dph when sex-specific genes showed differential expression.

No abstract

No abstract

Prolactin receptor (PRLR) and epidermal growth factor receptor (EGFR/ERBB) signaling pathways activated by prolactin (PRL) and epidermal growth factor (EGF), have a major role in the mammary gland development and in the etiology of breast cancer, respectively. ER+ breast tumors comprise up to 75% of all breast cancers and 10% of these are HER2+. Elevated levels of PRLR in breast tumors, high circulating levels of PRL and increased expression of ERBB1/2 in patients that become resistant to endocrine therapy have shown to be associated with higher risk of cancer progression. In this review, we examine the role of crosstalk between PRLR and ERBB1/2 signaling pathways in the activation of unliganded ERα, cyclin-D1 and other oncogenic factors (MYC, FOS, JUN) in breast cancer. PRL/PRLR and EGF/EGFR induces phosphorylation of ERα through activation of MEK/MAPK and PI3K/AKT signaling pathways. PRL in breast cancer cells via PRLR/JAK2 can also induce phosphorylation of ERBB2/HER2, which in turn activates the downstream RAS/MEK/ERK pathway required for ERα phosphorylation. EGFR, independent of PRL/PRLR, can activate STAT5 indirectly via c-SRC and drive the expression of target genes involved in cell proliferation and survival. The crosstalk between PRLR and HER2, where PRL induces HER2 signaling can be an alternative route for ERα activation to induce transcription of PRLR and other ER target genes. We believe that overexpression of EGFR/HER2 and PRLR in breast tumors could maximize the actions of their ligands, and further induce cell proliferation promoting malignancy. This could also explain the resistance to endocrine therapy resulting in tumor growth.