Increased frequency of CFTR gene mutations identified in Indian infertile men with non-CBAVD obstructive azoospermia and spermatogenic failure

High incidence of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene is associated with congenital bilateral absence of the vas deferens (CBAVD) and is considered as the genital form of cystic fibrosis (CF). The CFTR gene may also be involved in the etiology of male infertility in cases other than CBAVD. The present study was conducted to identify the spectrum and frequency of CFTR gene mutations in infertile Indian males with non-CBAVD obstructive azoospermia (n = 60) and spermatogenic failure (n = 150). Conspicuously higher frequency of heterozygote F508del mutation was detected in infertile males with non-CBAVD obstructive azoospermia (11.6%) and spermatogenic failure (7.3%). Homozygous IVS(8)-5T allele frequency was also significantly higher in both groups in comparison to those in normal healthy individuals. Two mutations in exon 25 viz., R1358I and K1351R were identified as novel mutations in patients with non-CBAVD obstructive azoospermia. Mutation R1358I was predicted as probably damaging CFTR mutation. This is the first report from the Indian population, emphasizing increased frequency of CFTR gene mutations in male infertility other than CBAVD. Thus, it is suggested that screening of CFTR gene mutations may be required in infertile Indian males with other forms of infertility apart from CBAVD and willing for assisted reproduction technology.     

Postmortem recovery and cryopreservation of spermatozoa from the vas deferens of rhesus macaques (Macaca mulatta)

To determine whether sperm derived from the vas deferens could be retrieved and successfully cryopreserved, testes were collected from 20 rhesus macaques (Macaca mulatta). The males ranged in age from 3 to 19 yr with an average age of 8.5 yr. No sperm was obtained from three animals that were younger than 4 yr. The remaining 17 samples contained sperm with an average sperm cell number of 421.8 ± 88.7 × 10⁶ and an average motility of 72.8 ± 4.4%. After 24 h of culture in TALP medium at 37 °C in 5% CO₂ and 95% air, the overall motility decreased significantly in all samples regardless of treatment. Freezing in TEST (TES-Tris buffer)-yolk buffer containing 6% (vol/vol) glycerol had a significant effect on sperm, reducing the immediate postthaw motility to 42.4% in nontreated samples. Treatment with dibutyryl-cAMP and caffeine further reduced sperm motility after 4 h in fresh sperm (72.8% vs. 50.4%) but increased motility in sperm that had been frozen (14.0% vs. 23.2%). The age of the male did not influence sperm concentration or grade but proved to be a significant factor in determining motility of frozen-thawed treated sperm, with lower motility found in samples from older males. Overall, the study demonstrates that motile sperm can be obtained from postmortem males, although subsequent studies will be needed to determine whether the quality is sufficient to facilitate its use in assisted reproduction.     

Foreign DNA introduced into the vas deferens is gained by mammalian spermatozoa

The uptake of exogenous DNA by mouse and rat spermatozoa was analyzed using in vitro and in vivo methods. Two DNA constructs were used, one containing the Growth hormone (GH) gene and the other the c-myc oncogene linked to the αA-crystallin promoter (CPV-1 plasmid). For the in vitro approach, washed epididymal spermatozoa were incubated for 2 hr in the presence of linearized DNA. For in vivo experiments, DNA was injected into the proximal region of the vas deferens, and spermatozoa were recovered 6 hr later. In situ hybridization employing fluorescent markers and electron microscopy were used to localize the exogenous genes in spermatozoa. The precise localization of the foreign DNA in spermatozoa was visualized by tridimensional reconstructions using a confocal laser microscopy. Uptake of exogenous DNA occurred in 60–70% of the spermatozoa after in vitro or in vivo treatments. A positive signal was detected in the sperm nucleus and was not affected by DNase treatments. Incorporation of exogenous DNA was also evaluated by slot blot and PCR techniques using the DNA isolated from the sperm nuclei and the corresponding labelled probes. Comparison of a nucleotide sequence between the DNA isolated from in vivo treated spermatozoa and CPV-1 plasmid showed a 98.6% identity. These results show the in vivo capacity of spermatozoa to incorporate exogenous DNA, the ability of this DNA to reach the nucleus, and also demonstrate that epididymal and vas deferens secretions do not block these capacities. Mol. Reprod. Dev. 51:42–52, 1998. © 1998 Wiley-Liss, Inc.     

49例先天性胎儿心脏疾病的静脉导管血流频谱分析

目的：不同的胎儿先天性心脏疾病通过不同的作用机制影响到胎儿心脏功能,会引起胎儿体内血循环的不同改变。静脉导管是胎儿血循环中重要的组成,也会随之出现相应的频谱改变。通过对49例合并先天性心脏疾病胎儿的静脉导管血流频谱及参数进行分析,研究胎儿不同类型心脏疾病对静脉导管（DV）血流频谱的影响。方法：选取2009年1月至2012年12月间我们在产前超声检查中发现的49例合并先天性心脏疾病的胎儿,分别测量DV血流频谱并进行参数分析,根据DV频谱是否正常分为两组。结果：DV频谱正常组有29例（59.18%）,表现为S波、a波的流速和方向正常,PVIV及DVRI指标位于正常范围。DV频谱异常组有20例,表现为S波流速降低、a波缺失或反向,PVIV及DVRI升高。结论：DV血流频谱和参数是评价胎儿心功能的良好指标。不同种类胎儿心脏发育异常对胎儿心功能影响的作用机制不同,其DV频谱也有着不同改变。通过对DV频谱的波形和参数分析,了解胎儿心脏异常的病生理机制,评价其严重程度和预后,这对于指导临床诊疗有着重要意义。     

Brain tissue transplanted to the anterior chamber of the eye: 2. Fluorescence histochemistry of immature catecholamine and 5-hydroxytryptamine neurons innervating the rat vas deferens

Summary Small pieces of the wall of the rat vas deferens were homologously transplanted to the anterior chamber of the eye together with small pieces of embryonic brain stem containing either developing noradrenaline (NA) cells of the locus coeruleus or 5-hydroxytryptamine (5-HT) neurons of the developing raphe system. The eyes of the recipients were sympathetically denervated. The double transplants became rapidly vascularized from the host iris. After 31/2 months the irides, together with their two transplants were analyzed by Falck-Hillarp fluorescence microscopy. Both the NA and the 5-HT neurons had survived and matured in the eye. Fluorescent varicose nerve terminals of the NA and 5-HT type respectively were found in all three potential receptor areas, i.e. within the CNS transplants, in the host irides and in the vas deferens transplants. In the latter, the newly formed monoamine nerve terminals arborized mainly within a well developed smooth muscle layer. The density of such new fibres was higher than or similar to that of the normally present sympathetic plexus in areas of the transplant close to the CNS transplant and lower in areas at a distance from the CNS transplant. It is concluded that immature central NA and 5-HT fibres are able to grow simultaneously into different types of sympathetically denervated smooth muscle tissues to form networks of fibres in the receptor organs resembling the normal sympathetic innervation.Supported by the Swedish Medical Research Council (04X-3185), and by grants from Magnus Bergvalls Stiftelse, Tidningen Expressens Prenatalforskningsfond and Karolinska Institutets fonder. We thank miss Ingrid Strömberg for skilful technical assistance. Nialamide^® was generously donated by Swedish Pfizer AB.     

胞外ATP在男性生殖道中的作用

胞外ATP除了能广泛作为神经递质外,还被认为是一种旁分泌或自分泌因子。ATP从男性生殖道中的精子或上皮细胞中释放,在调节各种生殖生理功能中起多种作用。本文综述了ATP调节附睾上皮细胞阴离子分泌的信号通路,阐述了ATP对依赖上皮细胞的输精管平滑肌收缩的调节机制,讨论了ATP在男性生殖道中的功能和作用。     

Effects of streptozotocin-induced diabetes and insulin on calcium responsiveness of the rat vas deferens

In the present study, effects of streptozotocin-induced diabetes and insulin treatment on the reactivity of rat vas deferens to KCl and calmidazolium, a calmodulin antagonist, were evaluated and calmodulin levels in vas deferens tissue from diabetic and insulin-treated rats were determined. Diabetes was induced in rats by a single injection of streptozotocin. Five weeks after the induction of diabetes, one group of diabetic rats was injected with insulin for 3 weeks. After 8 weeks, vas deferens tissues on one side of diabetic and insulin-treated diabetic rats and their controls were mounted in organ bath to measure isometric tension, while the tissues on the other side of rats were homogenized to determine calmodulin levels by radioimmunoassay. Concentration-response curves to KCl were obtained in vas deferens tissues in the absence and presence of calmidazolium. The effects of KCl and calmidazolium on vas deferens isolated from 8-weeks diabetic rats were decreased. Calmodulin levels were also found to be decreased in vas deferens from diabetic rats. Decreased calmodulin levels in diabetic rat vas deferens were not corrected by insulin treatment. Only a partial correction following insulin treatment was observed in contractile effect of KCl on diabetic rat vas deferens, whereas insulin treatment increases the affinity of calmodulin in this muscle. Experimental diabetes causes an impairment in calcium/calmodulin-dependent contractile process of vas deferens, which is correctable partially following insulin therapy. The changes in the function of rat vas deferens due to streptozotocin diabetes seem to be related to impaired sexual functions in human diabetes.     

Eine Untersuchung peripherer vegetativer Nervenfasern des Ductus deferens mit Hilfe der Gefrierätztechnik

Zusammenfassung Präterminale und terminale vegetative Nervenfasern aus dem Ductus deferens von Ratten und Mäusen wurden mit Hilfe der Gefrierätztechnik untersucht. Die Ultrastruktur der Axone in den Abdrücken stimmt mit dem Bild aus konventionell gewonnenen Präparationen im wesentlichen überein. Die Untersuchung ergab eine Zunahme membrangebundener Partikel in terminalen Abschnitten an der (+)-Seite des Axolemms. An freien Axolemmbereichen sowie an myoneuralen Kontakten treten diese Partikel, die einen Durchmesser zwischen 6–10 nm besitzen, in Form kleinflächiger Ansammlungen oder in größeren, plaqueförmigen Aggregationen auf. Membranfusionen von synaptischen Vesikeln mit dem terminalen Axolemm im Sinne eines herkömmlichen Exozytosevorganges konnten im untersuchten Material nicht nachgewiesen werden.

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A freeze-etching study of peripheral vegetative nerve fibres of the Ductus deferens

Summary Preterminal and terminal vegetative nerve fibres of the ductus deferens of rats and mice were studied with the freeze-etching technique. The ultrastructure of the axons in freeze-fractured and etched replicas is in agreement with that obtained by conventional electron microscopical techniques. An increase in the number of membrane-bound particles was observed in the terminal region of the (+)-side of the axolemma. These particles, wich have a diameter of 6–10 nm, tend to aggregate in either small or large, plaque-like accumulations at parts of the axolemmata, which are free from Schwann cells and at myoneural junctions. The possible function of these particle-aggregations is discussed. Fusions of synaptic vesicles with the terminal axolemma in the form of a conventional exocytotic process could not be detected in this material.

Mit Unterstützung durch den SFB 114 (Bionach).     

Effect of mating on sperm distribution in the reproductive tract of the male rat

Extragonadal sperm reserves in male rats were measured in different regions of the genital tract before and subsequent to normal ejaculation. In sexually rested rats, the sperm count (million spermatozoa for the paired organs) in different regions was: distal vas, 18; proximal vas, 9.8; cauda epididymidis, 229; caput + corpus epididymidis, 154. Following mating, the sperm count was reduced in the proximal and distal vas deferens and in the cauda epididymidis. The reproductive tract of mated females was found to contain 29% (no copulatory plug) or 59% (with copulatory plug) of the estimated mean ejaculate, which was estimated from the difference between the sperm counts in the sexually rested rat and following ejaculation. It is concluded that in the rat the immediate source of spermatozoa for ejaculation is the cauda epididymidis, with a smaller contribution arising from the vas deferens.