SummaryWe have generated a transgenic mouse line, Tg(Stra8-cre)1Reb (Stra8-cre), which expresses improved Cre recombinase under the control of a 1.4 Kb promoter region of the germ cell-specific stimulated by retinoic acid gene 8 (Stra8). cre is expressed only in males beginning at postnatal day (P)3 in early-stage spermatogonia, and is detected through pre-leptotene-stage spermatocytes. To further define when cre becomes active, we crossed Stra8-cre males with Tg(ACTB-Bgeo/ GFP)21Lbe (Z/EG) reporter females and compared the expression of Enhanced Green Fluorescent Protein (EGFP) with the protein encoded by the zinc finger and BTB domain containing 16 (Zbtb16) gene, PLZF -a marker for undifferentiated spermatogonia. Co-expression of EGFP is observed in the majority of PLZF+ cells. We also tested recombination efficiency by mating Stra8-cre;Z/EG males and females with wild-type mice and examining EGFP expression in the offspring. Recombination is detected in >95% of Z/EG+ pups born to Stra8-cre;Z/EG fathers but in none of the offspring born to transgenic mothers, a verification that cre is not functional in females. The postnatal, premeiotic, male germ cell-specific activity of Stra8-cre makes this mouse line a unique resource to study testicular germ cell development. KeywordsCre recombinase; Stra8 promoter; spermatogonia; spermatocytes; Z/EG These experiments were initiated to produce a transgenic mouse line that expresses cre in undifferentiated spermatogonia. To date, the use of cre-mediated recombination to inactivate genes in developing germ cells at specific stages has been limited by the restricted expression patterns of available cre drivers. Recombination in primordial germ cells is possible using the alkaline phosphatase, liver/bone/kidney Alpl tm1(cre)Nagy mouse line (Lomeli et al., 2000). However, significant developmental events occur between the onset of cre at embryonic day (E)9.5 and the appearance of spermatogonia in the postnatal testis. Additional cre activity is detected in the placenta, intestine, and neural tube. Excision of floxed DNA in primary spermatocytes and elongating spermatids is achievable using synaptonemal complex protein 1 Tg(Sycp1-cre)4Min (Vidal et al., 1998) and protamine 1 Tg(Prm-cre)58Og (O'Gorman et al., 1997), respectively, but the temporal expression of these transgenes is too late to be of use in undifferentiated spermatogonia. Meanwhile, the expression of cre driven by the growth differentiation factor 9 (Gdf9) and zona pellucida 3 (Lan et al., 2004;Lewandoski et al., 1997).We therefore selected the promoter of the premeiotic male and female germ cell-specific gene Stra8 to drive expression of improved Cre recombinase. Endogenous Stra8 is first expressed in ovarian germ cells at E12.5 and continues until E16.5, while in males it is first transcribed in early-stage spermatogonia in the postnatal testis and persists in premeiotic germ cells throughout adulthood (Menke et al., 2003;Oulad-Abdelghani et al., 1996). A 1.4 Kb Stra8 promoter fragment was recently fused to EGF...
Kisspeptin (Kiss1) and neurokinin B (NKB) (encoded by the Kiss1 and Tac2 genes, respectively) are indispensable for reproduction. In the female of many species, Kiss1 neurons in the arcuate nucleus (ARC) coexpress dynorphin A and NKB. Such cells have been termed Kiss1/NKB/Dynorphin (KNDy) neurons, which are thought to mediate the negative feedback regulation of GnRH/LH secretion by 17β-estradiol. However, we have less knowledge about the molecular physiology and regulation of Kiss1/Kiss1-expressing neurons in the ARC of the male. Our work focused on the adult male mouse, where we sought evidence for coexpression of these neuropeptides in cells in the ARC, assessed the role of Kiss1 neurons in negative feedback regulation of GnRH/LH secretion by testosterone (T), and investigated the action of NKB on KNDy and GnRH neurons. Results showed that 1) the mRNA encoding Kiss1, NKB, and dynorphin are coexpressed in neurons located in the ARC; 2) Kiss1 and dynorphin A mRNA are regulated by T through estrogen and androgen receptor-dependent pathways; 3) senktide, an agonist for the NKB receptor (neurokinin 3 receptor, encoded by Tacr3), stimulates gonadotropin secretion; 4) KNDy neurons express Tacr3, whereas GnRH neurons do not; and 5) senktide activates KNDy neurons but has no discernable effect on GnRH neurons. These observations corroborate the putative role for KNDy neurons in mediating the negative feedback effects of T on GnRH/LH secretion and provide evidence that NKB released from KNDy neurons is part of an auto-feedback loop that generates the pulsatile secretion of Kiss1 and GnRH in the male.
Gametes rely heavily on posttranscriptional control for their differentiation. Translational control, alternative splicing, and alternative processing of the 3' end of mRNAs are all common during spermatogenesis. Tenr, which encodes a highly conserved 72-kDa protein, is expressed solely in germ cells of the testis from the mid-pachytene stage until the elongating spermatid stage. TENR contains a double-stranded RNA binding domain, is localized to the nucleus, and is phylogenetically related to a family of adenosine deaminases involved in RNA editing. We show here that targeted mutation of the Tenr gene causes male sterility. Tenr mutant males have a reduced sperm count, and Tenr-/- sperm show a decrease in motility and an increase in malformed heads. Despite their sterility, some epididymal sperm from Tenr mutants have normal morphology. The ability of Tenr mutant sperm to fertilize zona pellucida-free oocytes and to bind to, but not fertilize, zona pellucida-intact oocytes suggests that the normal-appearing sperm are not able to penetrate the zona pellucida. These data suggest that TENR plays an essential function in spermatid morphogenesis.
IntroductionT cells play an important role in the pathogenesis of systemic lupus erythematosus (SLE). Clonal expansion of T cells correlating with disease activity has been observed in peripheral blood (PB) of SLE subjects. Recently, next-generation sequencing (NGS) of the T cell receptor (TCR) β loci has emerged as a sensitive way to measure the T cell repertoire. In this study, we utilized NGS to assess whether changes in T cell repertoire diversity in PB of SLE patients correlate with or predict changes in disease activity.MethodsTotal RNA was isolated from the PB of 11 SLE patients. Each subject had three samples, collected at periods of clinical quiescence and at a flare. Twelve age-matched healthy controls (HC) were used for reference. NGS was used to profile the complementarity-determining region 3 (CDR3) of the rearranged TCR β loci.ResultsRelative to the HC, SLE patients (at quiescence) demonstrated a 2.2-fold reduction in repertoire diversity in a given PB volume (P <0.0002), a more uneven distribution of the repertoire (Gini coefficient, HC vs SLE, P = 0.015), and a trend toward increased percentage of expanded clones in the repertoire (clone size >1.0 %, HC vs SLE, P = 0.078). No significant correlation between the overall repertoire diversity and clinical disease activity was observed for most SLE patients with only two of eleven SLE patients showing a decreasing trend in repertoire diversity approaching the flare time point. We did not observe any overlap of CDR3 amino acid sequences or a preferential Vβ or Jβ gene usage among the top 100 expanded clones from all SLE patients. In both HC and SLE, the majority of the expanded clones were remarkably stable over time (HC = 5.5 ±0.5 months, SLE = 7.2 ±2.4 months).ConclusionsA significant decrease in T cell repertoire diversity was observed in PB of SLE patients compared to HC. However, in most SLE patients, repertoire diversity did not change significantly with increases in disease activity to a flare. Thus, without a priori knowledge of disease-specific clones, monitoring TCR repertoire in PB from SLE patients is not likely to be useful to predict changes in disease activity.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0655-9) contains supplementary material, which is available to authorized users.
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