Porcine CPEB1 is involved in Cyclin B translation and meiotic resumption in porcine oocytes

Ovarian immature oocytes accumulate many dormant maternal mRNAs, which have short poly(A) tails. Cytoplasmic‐polyadenylation‐element binding protein (CPEB) has been reported to play key roles for the elongation of the tails and the translation of these mRNAs in Xenopus oocytes. However, the functions of CPEB in meiotic resumption have not yet been established in mammalian oocytes. The present study examined the roles of porcine CPEB in Cyclin B syntheses and meiotic resumption of porcine oocytes. Porcine CPEB1 (pCPEB1) cDNA was cloned from total RNA of immature oocytes by RT‐PCR. The overexpression of pCPEB1 by mRNA injection into immature oocytes increased Cyclin B expression and the rate of meiotic resumption. Conversely, the inhibition of endogenous CPEB by expression of a dominant‐negative mutant pCPEB1 (AA‐CPEB), which replaced the expected phosphorylation sites with alanines, had the effect of inhibiting Cyclin B synthesis, ribosomal S6 kinase phosphorylation (an indicator of Mos activity), and meiotic resumption. The inhibition of porcine Aurora A by an injection of antisense RNA enhanced the inhibitory effects of AA‐CPEB. These results suggest the involvement of mammalian CPEB1 in Cyclin B syntheses and meiotic resumption in mammalian oocytes. In addition, the phosphorylation sites of pCPEB1 were identified and are suggested to be phosphorylated by porcine Aurora A.     

Expression of a testis-specific form of TBP-related factor 2 (TRF2) mRNA during mouse spermatogenesis

The gene encoding TATA-binding protein-related factor 2 (TRF2/TLF/TLP/TRP), essential for the progress of spermiogenesis, is abundantly expressed in mammalian testis. A sequence database search revealed that mouse TRF2 is encoded by two mRNAs containing the same protein-coding region and different 5'-untranslated regions. Northern blot analysis using DNA probes specific for the 5'-untranslated regions demonstrated that these two mRNAs are distinguished from each other by the expression patterns: ubiquitous and testis-specific expression. The ubiquitously expressed form of TRF2 mRNA was present at a very low level throughout testicular development, whereas expression of the testis-specific form was first detectable in the 14-day-old testis, and the mRNA level abundantly increased at the later stages of testicular development. Western blot analysis indicated that the TRF2 level increases during testicular development, which is consistent with the expression pattern of the testicular form of TRF2 mRNA. Thus, the presence of the testis-specific form of TRF2 mRNA may account for overexpression of the TRF2 gene in the testis.     

The I510V mutation in KLHL10 in a patient with oligoasthenoteratozoospermia

Oligoasthenoteratozoospermia is a human infertility syndrome caused by defects in spermatogenesis, spermiogenesis, and sperm maturation, and its etiology remains unclear. Kelch-like 10 (KLHL10) is a component of ubiquitin ligase E3 10 (KLHL10) and plays an important role in male fertility. Deletion or mutation of the Klhl10 gene in Drosophila or mice results in defects in spermatogenesis or sperm maturation. However, the molecular mechanisms by which KLHL10 functions remain elusive. In this study, we identified a missense mutation (c.1528A→G, p.I510V) in exon 5 of KLHL10, which is associated with oligoasthenoteratozoospermia in humans. To investigate the effects of this mutation on KLHL10 function and spermatogenesis and/or spermiogenesis, we generated mutant mice duplicating the amino acid conversion using the clustered regularly interspaced palindromic repeat/caspase 9 (CRISPR/Cas9) system and designated them Klhl10^I510V mice. However, the Klhl10^I510V mice did not exhibit any defects in testis development, spermatogenesis, or sperm motility at ten-weeks-of-age, suggesting that this mutation does not disrupt the KLHL10 function, and may not be the cause of male infertility in the affected individual with oligoasthenoteratozoospermia.     

Effects of vinclozolin administration on sperm production and testosterone biosynthetic pathway in adult male rat

The effect of vinclozolin (VCZ), used as a fungicide and known to have anti-androgenic effects on spermatogenesis and gene expression in the male rat testis was investigated. In Experiment 1, VCZ (100 mg/kg/day) or flutamide (FM, 25 mg/kg/day) was orally administered to male Holzman rats for six days. 8 days after the last administration (D8), a drastic increase in intratesticular testosterone was detected in FM (4.2-fold over control) but not in VCZ treated animals, whereas on D36 post-administration, both groups showed similar levels. Significant decreases in daily sperm production were seen in both VCZ and FM-treated rats on D36. Semiquantitative RT-PCR analysis with testicular and pituitary mRNAs on D8 revealed that LHbeta and FSHbeta mRNAs were increased in the pituitary by VCZ, as well as by FM. Among the four testicular steroidogenic enzyme genes, cytochrome P450 side chain cleavage (P450scc) and cytochrome P450 17alpha/C(17-20) lyase (P450c17) mRNAs were significantly increased, whereas 17beta-hydroxysteroid dehydrogenase type III (17betaHSD) mRNA was not changed. A significant increase in 3beta-hydroxysteroid dehydrogenase type I (3betaHSD) and a decrease in androgen receptor (AR) mRNA were observed only in FM treated rats. Immunohistochemistry demonstrated intense staining of P450scc in the interstitial cells of VCZ-treated testis on D8. In Experiment 2, hormone levels were measured at 1, 3, 6, 12 and 24 hours after VCZ (100 mg/kg) administration to Sprague-Dawley rats. Serum LH level remained constant for the first 3 hours and started to increase at 6 hrs. In contrast, serum and intratesticular testosterone levels increased 2-fold at 1 hr and maintained the level until 24 hrs. P450c17 mRNA level was 2-fold increased at all periods, whereas no obvious changes were detected in the other steroidogenic enzyme genes. Although not statistically significant, AR mRNA level increased 2-fold, 3 hrs after VCZ administration. These results indicate that VCZ affects the pituitary in a similar manner as FM, but functions differently on testicular gene expression.     

miRNA在哺乳动物配子发生中的作用研究进展

哺乳动物配子发生的基因表达调控包括编码基因的阶段特异性表达调控及非编码基因的转录和转录后水平调控。微小RNA(microRNA, miRNA)作为一类小的非编码RNA, 通过识别靶基因非翻译区的结合位点, 导致mRNA降解或者蛋白质翻译抑制, 从而在转录后水平发挥调控作用。近年来, miRNA在哺乳动物生殖活动中的作用逐渐被揭示, 越来越多的研究表明, miRNA在哺乳动物精子发生、精子成熟、卵母细胞成熟、卵泡发育及早期胚胎发育等过程中都发挥着重要的调节作用, 其可通过调节支持细胞的增殖和凋亡或精原细胞、精母细胞及精细胞的细胞周期进程, 在精子发生的不同阶段发挥间接或直接调控作用, 也可通过调节卵母细胞、卵丘细胞以及颗粒细胞的增殖、凋亡、激素合成和细胞间作用, 对卵母细胞的发育和成熟过程进行调控。作者主要介绍了在哺乳动物配子发生过程中, miRNA的细胞和阶段特异性表达及其对靶基因的调节作用, 以期为深入研究哺乳动物配子发生的调控机制提供参考。     

Alterations of gene expression in adult male rat testis and pituitary shortly after subacute administration of the antiandrogen flutamide

In the course of profiling alterations of gene expression in the male reproductive system induced by anti-androgenic agents, 28 genes expressed in the testis or pituitary of adult rats were examined shortly after subacute administration of the well-known anti-androgen, flutamide (FM). FM (25 mg/kg/day) was orally administered to male rats for six days. On day 8 (D8) after the first dose of FM, intratesticular testosterone (T) levels had dramatically increased, but daily sperm production on D36 was significantly decreased. The mRNA levels of testicular and pituitary genes on D8 were measured by semiquantitative RT-PCR. Among the six testicular steroidogenic enzyme genes, the mRNAs of the P450 side chain cleavage, P450 17 alpha/C(17-20) lyase, and 3beta-hydroxysteroid dehydrogenase type I (3betaHSD) genes significantly increased, whereas 17beta-hydroxysteroid dehydrogenase type III slightly decreased. Among the three steroid receptors examined, androgen receptor (AR) and glucocorticoid receptor (GR) mRNAs were significantly down-regulated (29% and 35%, respectively) in the testis, but there was no change in estrogen receptor alpha. There were no clear changes in expression of the gonadotropin receptors and Sertoli cell specific genes, but a slight increase was observed in expression of the lactose dehydrogenase-c mRNA, a germ cell specific gene. Among the three immediate early genes, c-myc mRNA was increased approximately 1.4-fold. In the pituitary, on the other hand, mRNAs for LHbeta and FSHbeta subunits and gonadotropin releasing hormone receptor had increased significantly. These results show that subacute FM administration first affected hypothalamus/pituitary hormone gene expression, then altered gonadotropin secretion, and subsequently induced over-expression of testicular steroidogenic enzyme genes. However, the significant up-regulation of 3betaHSD and down-regulation of AR mRNAs, despite the higher level of intratesticular T, might be explained by an antagonistic action of hydroxyflutamide retained in the testis. The profiles of alterations in gene expression observed will provide important information for the screening of adult male animals for anti-androgenic chemicals.     

Expression of CYP26b1 and Related Retinoic Acid Signalling Molecules in Young,Peripubertal and Adult Dog Testis

The objective of the study was to elucidate mRNA expression of CYP26b1 (cytochrome P450, family 26, subfamily B, polypeptide 1) and signalling molecules ALDH1 (aldehyde dehydrogenase 1), CRABPII (cellular retinoic acid‐binding protein II), RARα (retinoic acid receptor alpha) and STRA8 (stimulated by retinoic acid gene 8) in dog testis from different post‐natal developmental ages. Testicular tissue samples were collected from medium‐sized mixed breed dogs at different ages such as young (<4 months; N = 4), peripubertal (4–8 months; N = 3) and adult (>8 months; N = 4) were used to evaluate relative mRNA expression. Genes of RA‐degrading enzyme CYP26b1, ALDH1 involved in RA synthesis and genes of carrier protein CRABPII involved in RA metabolism were turned on during the post‐natal testicular development in dogs. Their expression pattern differs at different developmental ages (p < 0.05), and the levels of mRNA expression were compensated towards a normal developmental response for the sexual maturity and continuous spermatogenesis. The mRNA expression of RARα, one of the RA receptors participates in RA signalling in connection to spermatogenesis, was recorded in young and adult stages at varying degree. STRA8 is one of the responsive genes with regard to meiosis, and this functional gene product was expressed in all ages with the changing level (p < 0.01). In summary, the expression pattern of RA signalling molecules differed from young to adult ages, and it is expected that these changes are to compensate towards a normal developmental response for the sexual maturity and continuous spermatogenesis.     

Effects of oral exposure of bisphenol A on mRNA expression of nuclear receptors in murine placentae assessed by DNA microarray

Bisphenol A (BPA), a candidate endocrine disruptor (ED), is considered to bind to estrogen receptors and to regulate expressions of estrogen responsive genes. It has also shown evidence of affecting the reproductive, immunological and nervous systems of mammalian embryos. However, the effects of BPA on placentae, a central organ of feto-maternal interlocution, are still unclear. To reveal the mechanisms of BPA effects on placentae in mammals, we compared the mRNA expression of 20 nuclear receptors between placentae of vehicle controls and those of orally BPA exposed pregnant mice by a DNA microarray technique. In murine placentae, mRNAs of 11 nuclear receptors were not detected. However, greater than 1.5 fold changes in mRNA expression of nine nuclear receptors between vehicle control and BPA treated mice were noted. Moreover, remarkable changes in mRNA expression of six non-nuclear receptor proteins were induced by BPA exposure. There were various differences in the effects of BPA on the expression of these mRNAs between the placentae with male embryos and those with female embryos. Such embryo-sex dependent differences are interesting and important pointers to understanding of the endocrine disrupting effect of BPA. The present data indicate that BPA affects the expression of nuclear receptor mRNAs in placentae and may disrupt the physiological functions of placentae.     

A new ENU-induced mutant mouse with defective spermatogenesis caused by a nonsense mutation of the syntaxin 2/epimorphin (Stx2/Epim) gene

Repro34 is an N-ethyl-N-nitrosourea (ENU)-induced mutation in mice showing male-specific infertility caused by defective spermatogenesis. In the present study, we investigated pathogenesis and molecular lesions in relation to spermatogenesis in the repro34/repro34 homozygous mouse. Histological examination of the testis showed that the seminiferous epithelium of the repro34/repro34 mouse contained spermatogonia and spermatocytes but no round and elongating spermatids. Instead of these haploid cells, multinucleated giant cells occupied the niche of the seminiferous tubules. Immunohistochemical staining for Hsc70t, an elongating spermatid specific protein, confirmed the absence of elongating spermatids. Furthermore, RT-PCR showed that there were significantly reduced expressions of the marker genes specifically expressed in the spermatid and that there was no difference in the expressions of the spermatocyte specific marker genes. These findings indicated interruption of the spermatogenesis during transition from the spermatocyte to spermatid in the repro34/repro34 mouse. The repro34 locus has been mapped on a 7.0-Mb region of mouse chromosome 5 containing the Syntaxin 2/Epimorphin (Stx2/Epim) gene, and targeted disruption of this gene has been reported to cause defective spermatogenesis. We therefore sequenced the entire coding region of the Stx2/Epim gene and found a nucleotide substitution that results in a nonsense mutation of this gene. The expression pattern of the Stx2/Epim gene during the first wave of spermatogenesis, increased expression at later stages of spermatogenesis, was in agreement with the affected phase of spermatogenesis in the adult repro34/repro34 testis. We therefore concluded that the male infertility of the repro34/repro34 mouse is caused by the interruption of spermatogenesis during transition from the spermatocyte to spermatid and that the nonsense mutation of the Stx2/Epim gene is responsible for the interruption of spermatogenesis.     

小鼠实验性隐睾诱发生殖细胞类型变化

利用 3 0～ 3 5日龄昆白系小鼠制作实验性隐睾 ,定期分批朴杀取样 ,检查隐睾组织学及生殖细胞群体变化 ,为生殖细胞富集及提高体内精原干细胞转基因效率提供条件和依据。结果表明 ,盆腔隐睾精子发生被阻断于精子形成阶段 ;经历 1 5d以上 ,曲细精管内精子数量较少 ;腹腔隐睾精子发生被阻断于精原细胞向精母细胞过渡阶段 ;经历 3 0 d以上 ,曲细精管仅由精原细胞、少量精母细胞及支持细胞组成。由此可知 ,制作盆腔隐睾 ,可得到含少量精子的生殖细胞群体以及主要含精原细胞的生殖细胞群体