Fine structure and FSH binding of Sertoli cells in the blue fox (Alopex lagopus) in different stages of reproductive activity

A clearly different Sertoli cell morphology was found in the immature blue fox and in the adults in and out of season. Immature cells had small, basally, located nuclei rich in peripheral heterochromatin, and few cytoplasmic organelles. Sertoli cells from adults in season had basally located, large, convoluted nuclei with homogenous nucleoplasma and prominent nucleoli. The basal and intermediate parts of the cytoplasm contained an extensively developed ER, numerous mitochondria, free ribosomes, lipid droplets and residual bodies, while the apical cytoplasm showed few distinct structures. In Sertoii cells from adults out of season the nuclei were dislocated towards the lumen, and apart from numerous dilated cisternae of ER, there were generally fewer organelles, but more glycogen particles. A marked rise in FSH binding was found towards the breeding season.     

Seasonal changes in testicular structure and function in the blue fox (Alopex lagopus), as quantified by morphometric analysis and measurement of adenylate cyclase activity

The volume of the blue fox testis showed 5-fold changes during the year, associated with considerable changes in cellular composition. The seminiferous epithelium was maximally regressed in August, when 94% of tubules contained only spermatogonia. By late October, approximately 6 months before the mating season, 40% of tubules contained primary spermatocytes. From the middle of January until the end of April all tubules contained spermatids or more advanced haploid cells. Tubular diameter increased by 73% during testicular re-development, and epithelial height increased 3-fold. Regression to the basal state occurred during May to July. The volume densities of the seminiferous epithelium and of interstitial tissue remained approximately constant throughout the year. Soluble Mn2+-dependent adenylate cyclase activity showed seasonal variations that paralleled those of the haploid germ cell population and testicular volume, whereas somatic cell adenylate cyclase activity was relatively constant.     

The effect of gossypol acetic acid on the different stages of the spermatogenic cycle in the rat

The reversibility of the effect of gossypol on testicular histology and fertility was studied in rats. Adult males of proven fertility were treated orally with gossypol acetic acid (15 mg/kg) for 9 or 16 weeks (groups 1 and 2, respectively). Another groups of animals (group 3) was given gossypol (15 mg/kg) for 16 weeks and killed 6 weeks after the end of treatment. Control animals (group 4) were given the vehicle only by oral intubation. In the mating studies, although only 33% of the animals in group 1 were infertile, 100% infertility was observed following 16 weeks of gossypol treatment (group 2). All animals in group 3 regained their fertility 6 weeks after cessation of drug treatment. Damage was observed to 15.7% of the seminiferous tubules after 9 weeks of drug treatment, and to 78% after 16 weeks of treatment. Extensive vacuolization, increased numbers of lipid droplets, degeneration of germ cells, loosening of the epithelium, and a significant decrease in the number of pachytene spermatocytes (stages VII-X) and spermatids (steps 7-10 at stages VII-X) were observed after gossypol treatment. There was a decrease in the diameter of only stage VIII seminiferous tubules after 9 weeks of treatment, whereas a reduction was observed in the tubules of all stages after 16 weeks of gossypol treatment. In the recovery phase, the diameter of seminiferous tubules was similar to that of controls, except for tubules at stage VIII. No change in the area of the lumen of the seminiferous tubules and lipid bodies was observed after 9 weeks of drug treatment, but a marked reduction in the area of the lumen (stages II-X) and an increase in lipid bodies (all stages) was observed after 16 weeks of gossypol treatment. Six weeks after cessation of treatment, the area of the lumen and the number of lipid bodies were comparable to values in controls. A reduction in the area of the epithelium was restricted to just a few stages (VIII-XIV) in treated animals at 9 weeks, whereas after 16 weeks the area of the epithelium was decreased in all tubules. In the recovery phase, except for tubules at stage VIII, the area of the seminiferous epithelium was comparable to that in controls.     

Effect of Black maca (Lepidium meyenii) on one spermatogenic cycle in rats

Lepidium meyenii (Maca) grows exclusively between 4000 and 4500 m above sea level in the Peruvian central Andes. The hypocotyls of this plant are traditionally used in the Andean region for their supposed fertility-enhancing properties. The hypocotyls have different colours. Of these, Black maca has better effects on spermatogenesis. The present study aimed to test the hypothesis that Black maca has early effects during a spermatogenic cycle (12 days) of male rats. For this, testicular spermatid, epididymal sperm and vas deferens sperm counts were measured after 1, 3, 5, 7 and 12 days of treatment with Black maca. Aqueous extract of Black maca was given orally by daily gavage at a dose of 2 g kg(-1). In a spermatogenic cycle, compared with day 1, daily sperm production (DSP) was lower at day 7 (control), whereas with Black maca, the difference was observed at day 12. Epididymal sperm count was higher in rats treated with Black maca at days 1, 3 and 7, but similar to controls at days 5 and 12; similarly sperm counts in vas deferens was higher in rats treated with Black maca in days 3, 5 and 7, but similar to controls at days 1 and 12. From this, it is suggested that first action of Black maca was at epididymal level increasing sperm count after 1 day of treatment, whereas an increase in sperm count was observed in vas deferens at day 3 of treatment. Finally, an increase in DSP was observed after 7 days of treatment with Black maca. Testicular testosterone was not affected after 7 days treatment with Black maca. In conclusion, Black maca affects sperm count as early as 1 day after beginning of treatment.     

Seasonal Change in Fine Structure and Function of Leydig Cells in the Blue Fox (Alopex lagopus)

The correlation between ultrastructural alterations and presumptive change in endocrine activity was studied in the Leydig cells of 17 blue foxes castrated at different times of the year.
In the reproductive season (March and April), with high concentrations of plasma testosterone and very active spermatogenesis the Leydig cells had large and light nuclei, few lipid droplets, ovoid mitochondria with tubular cristae, and a well developed agranular endoplasmic reticulum (AER). During early regression the mitochondria became large and pleomorphic, and the AER was arranged in concentric whorls. Later, when the activity seemed to have reached basal levels, the nuclei were small and dark, the number of lipid droplets increased, the mitochondria were rod-shaped with lamellar cristae, and the whorls of AER decreased. During the period of increasing activity the nuclei enlarged, the endoplasmic reticulum displayed both granular and agranular profiles, the mitochondria were sometimes dark and cup-shaped, and the number of lipid droplets decreased gradually.     

Soluble Mn2+-dependent adenylate cyclase activity in the testis of the blue fox (Alopex lagopus)

Soluble Mn2+-dependent adenylate cyclase (MnAC) activity was found in testicular cytosol from blue foxes castrated during the breeding season. The rate of MnAC activity was approximately constant for 30 min at 35 degrees C and for 2 hr after storage at 25 degrees C. Activity was directly proportional to cytosol protein concentration and was optimal in the physiological pH range. Enzyme activity declined in the presence of an alkylating agent (N-ethyl maleimide, NEM) and was eliminated at a concentration of 1 mM NEM. Low concentrations (0.1-10 mM) of a reducing agent (beta-mercapto ethanol, beta ME) did not increase MnAC activity, whereas a high concentration (100 mM) led to a significant reduction (p less than 0.01) in activity. Substitution of Mn2+ in the assay medium with Mg2+ led to a total loss of enzyme activity, which could not be regained by adding hormones or by preincubation of cytosol for 60 min. The Km for Mn2+ was estimated to be 3.5 mM. The affinity of the enzyme for Mn2+ was not altered by varying the concentration of ATP. In contrast, increasing concentrations of Mn2+ appeared to increase the affinity of the enzyme for MnATP2-. The Km for MnATP2- thus varied from 6 to 18 mM.     

Seasonal changes in spermatogenic activity and in plasma levels of FSH, LH and testosterone, and the effect of immunization against inhibin in the male silver fox (Vulpes vulpes)

The cellular composition of the silver fox testis assessed by DNA flow cytometry and histological analysis exhibited marked circannual alterations. The proportion of haploid cells increased from late October to the breeding season in February, while that of diploid cells decreased and that of tetraploid cells fluctuated during the same period. Towards the end of March these changes were reversed. The seasonal variations in testicular histology paralleled the changes in distribution of cells from the different DNA populations. In August, 69% of the tubules contained spermatogonia as the only type of germ cell, while the remaining 31% also contained a few primary spermatocytes. In late October more than 50% of the tubules contained spermatocytes, and during the period of further activation from early December-February the seminiferous epithelium included round and/or elongated spermatids as well. In February, all tubules contained complete associations of germ cells, whereas in late March tubules with spermatogonia only and spermatogonia together with a few spermatocytes reappeared. In May, only such tubules could be found indicating total regression. Plasma concentrations of FSH and LH increased from early November, both gonadotrophins reaching maximum levels in December or early January, and then both declined during the second part of January, immediately prior to the actual breeding season. LH values showed a few smaller peaks in the beginning of June, whereas FSH levels were generally low until the next period of testicular reactivation. Testosterone concentrations were also low during most of the year but rose in November and December to reach a peak in January and a second peak in June. In animals immunized against inhibin the distribution of haploid, diploid and tetraploid cells did not deviate to any great extent from that in the controls, except in March when the immunized males had a markedly lower proportion of tetraploid cells, and in May, when they had a distinctly higher proportion of haploid cells. These findings were partly reflected by the histology. In the immunized animals, plasma FSH levels started to increase at approximately the same time but peaked higher and remained elevated almost 1 month longer than in the controls, whereas both the rise and decline in LH levels generally coincided with the variations in these animals, but the values were mostly higher. The testosterone profiles were similar to those in the controls except that the maximum values were also usually higher.     

LM23 is a novel member of the Speedy/Ringo family at the crossroads of life and death of spermatogenic cell

LM23 is a gene specifically expressed in the testis of Rattus norvegicus, as previously reported by our laboratory. The aim of the study is to further investigate the biological function of LM23. Several bioinformatic tools were utilized, including PROSITE and BLAST. To determine the subcellullar localization of LM23, a polyclonal antibody specific for LM23 was generated via the immunization of rabbits. The LM23 gene was cloned from rat testis tissue, and LM23 protein was expressed in Escherichia coli. The biological function of LM23 was analyzed with microarray analysis and immunohistochemistry, using a rat model of LM23 gene knockdown. The results suggested that LM23 belongs to the Speedy/Ringo family. LM23 regulated the G₁/S and G₂/M transitions of the cell cycle during spermatogenesis. Downregulation of the LM23 gene during spermatogenesis could lead to the activation of both the Fas–FasL pathway and the mitochondrial pathway. These novel findings indicate that LM23 has a diverse array of functions that are important in both the life and death of the spermatogenic cell.     

Age-dependent expression of the cystatin-related epididymal spermatogenic （Cres） gene in mouse testis and epididymis

Aim： To investigate the spatial and temporal expression of the cystatin-related epididymal spermatogenic （Cres） gene in mouse testis and epididymis during postnatal development. Methods： The QuantiGene assay and indirect immunofluorescence technique were used to examine the Cres mRNA and Cres protein level in mouse testis and epididymis on postnatal days 14, 20, 22, 28, 35, 49, 70 and 420. Results： （1） In both the testis and epididymis, Cres mRNA was fast detected on day 20, then it increased gradually from day 20 to day 70, and the high expression level maintained till day 420. （2） In the testis, the Cres protein was exclusively localized to the elongating spermatids and was first detected on day 22. The number of Cres-positive spermatids increased progressively till day 49. From day 49 to day 420, the number of Cres-positive cells was almost stable. （3） The Cres protein was first detected on day 20 in the proximal caput epididymal epithelium. By day 35, the expression level of the Cres protein increased dramatically and the high level was maintained till day 420. Moreover, the luminal fluid of the midcaput epididymis was also stained Cres-positive from day 35 on. No Cres-positive staining was observed in distal caput, corpus and cauda epididymis throughout. Conclusion： The Cres gene displays a specific age-dependent expression pattern in mouse testis and epididymis on both the mRNA and protein level.     

Quantitative （stereological） study of incomplete spermatogenic suppression induced by testosterone undecanoate injection in rats

Aim: To evaluate the key lesions in spermatogenesis suppressed partially by testosterone undecanoate(TU) treatment. Methods: Adult male SD rats were treated with vehicle or TU (19 mg/kg) injection (i.m.) every 15 days for 130 days. The numbers of all types of cells (nuclei) in the seminiferous tubules and the interstitial tissue were estimated using a contemporary stereological tool, the optical disector. Results: In response to TU treatment, the numbers of non-type B spermatogonia, type B sperrnatogonia and late elongated spermatids per testis were reduced to 51%, 66% and 14% of the controls, respectively. The conversion ratios from type B spermatogonia to early spermatocytes and pachytene spermatocytes were not significantly affected and the ratios to the later germ cell types fell to 51%-65% of the controls. Less than 1.0 % of immature round spermatids were seen sloughing into the tubule lumen, 4.0% of elongated spermatids retained in the seminiferous epithelium, and about half of the elongated spermatid nuclei appreciably malformed. Leydig cells were atrophied but their number and the peritubular myoid cell number per testis were unchanged. Conclusion: Double inhibition of spermatogenesis (i.e. inhibition at spermiation and spermatogonial conversion to type B spermatogonia), a scenario seen in the monkey and human following gonadotrophin withdrawal, was not sufficiently effective for a complete spermatogenic suppression in the rat after TU treatment, probably due to ineffective inhibition of the Leydig cell population and therefore the intra-testicular test-osterone levels. (Asian J Androl 2004 Dec; 6: 291-297)     

The A-type cyclins and the meiotic cell cycle in mammalian male germ cells

There are two mammalian A-type cyclins, cyclin Al and A2. While cyclin A1 is limited to male germ cells, cyclin A2 is widely expressed. Cyclin A2 promotes both Gl/S and G2/M transitions in somatic cells and cyclin A2-deficient mice are early embryonic lethal. We have shown that cyclin Al is essential for passage of spermatocytes into meiosis I (MI) by generating mice null for the cyclin A1 gene Ccna1. Both Ccna1(-/-) males and females were healthy but the males were sterile because of a cell cycle arrest before MI. This arrest was associated with desynapsis abnormalities, low M-phase promoting factor activity, and apoptosis. We have now determined that human cyclin A1 is expressed in similar stages of spermatogenesis and are exploring its role in human male infertility and whether it may be a novel target for new approaches for male contraception.     

瘢痕疙瘩不同部位P_(53)蛋白的表达和细胞周期的分布

目的　探讨瘢痕疙瘩周边部和中央部细胞生物学差异以解释瘢痕疙瘩呈浸润性生长的机理。方法　取手术切除的瘢痕疙瘩组织各 6例为标本 ,通过细胞培养 6～ 10代后 ,应用流式细胞仪、粘附式细胞仪检测瘢痕疙瘩不同部位成纤维细胞P53 蛋白的表达和细胞周期的分布。结果　瘢痕疙瘩周边部成纤维细胞大量分布于增殖期 (G2 、S、M期 ) ,且P53 蛋白表达较低 ;而瘢痕疙瘩中央部成纤维细胞P53 蛋白呈强表达 ,且主要分布在静止期 (G1、G0 期 )。结论　瘢痕疙瘩不同部位成纤维细胞细胞周期的分布及P53 蛋白表达的差异可能是导致其不同生长特性的细胞生物学机理之一 ,也可能是导致瘢痕疙瘩呈浸润性生长的机理之一。     

红蓝光交替照射联合药物治疗痤疮疗效观察

目的:观察红蓝光交替照射联合药物治疗痤疮的疗效和安全性。方法:将118例轻、中度痤疮患者随机分为实验组和对照组。实验组采用红蓝光交替照射联合本院自制的痤疮灵洗剂治疗;对照组仅用痤疮灵洗剂治疗。采用痤疮综合分级系统评价治疗前后疗效,同时观察不良反应。结果:实验组和对照组有效率分别为83.05%和44.07%,两组有效率存在显著性差异(χ2=4.476,P=0.000);治疗结束时两组痤疮综合分级系统评分均较治疗前下降,实验组明显低于对照组(P<0.05),两组均无明显不良反应。结论:红蓝光交替照射联合药物治疗痤疮安全、有效。     

红蓝光治疗痤疮临床疗效观察

目的:探讨红蓝光照射治疗痤疮的临床疗效和安全性。方法:将113例轻、中度痤疮患者分为两组,实验组采用本院自制的克林霉素醇溶液联合红蓝光照射治疗,对照组仅外用本院自制的克林霉素醇溶液治疗。采用痤疮综合分级系统(GAGS)评价治疗前、后疗效。结果:实验组和对照组有效率分别为82.8%和43.6%,两组的治疗有效率存在显著性差异(P<0.01)。GAGS综合分值均较治疗前明显下降(P<0.01),但实验组下降更显著。结论:红蓝光治疗面部轻、中度痤疮安全有效。     

The role of Intraoperative cholangiography (IOC) and methylene blue tests in reducing bile leakage after living donor hepatectomy

AimLiver transplantation remains the only curative treatment in end stage liver disease. Biliary complications remain the most common major morbidity causes in hepatic resection. We aimed to determine and eliminate the bile leakage in donor hepatectomy of LDLT.Material and methodsThis study was conducted retrospective and one center study. The study population included 110 consequential liver donors with major liver resection (more than three segments). The population was divided into three groups for data analyses. Primary study groups included 40 donors subjected to methylene blue test starting in April 2013 and 40 donors subjected to intraoperative cholangiography started in March 2014.ResultsA total of 110 liver donors (42.7% women) were included in the study. Postoperative biliary complications were less in methylene blue and intraoperative cholangiography (IOC) groups. Bile leakage was significantly higher in control group (23.3%) compared to methylene blue (5%) and IOC groups (2%) Average duration of hospital stay and duration of operation were significantly higher in control group compared to methylene blue and IOC groups.ConclusionIn our study we conducted to establish biliary leakage in living donor hepatectomy which intraoperative cholangiography test was used to determine. Many intraoperative methods have been introduced to prevent biliary leakage and development of complications. We have showed that IOC test used in the present study could be easily applied in both living liver donor hepatectomy and other major hepatectomy cases. IOC test reduced postoperative biliary leakage incidence and did not increase incidence of other complications.     

The value and role of low dose methylene blue in the surgical management of hyperparathyroidism

Introduction

Methylene blue (MB) has been used in the identification of abnormal parathyroid glands in surgery for hyperparathyroidism. Its efficacy and safety profile have been questioned recently and this study sought to demonstrate such aspects in a unit where its use is routine.

Methods

Prospective data collected over six years in a single surgeon’s practice were interrogated to identify factors affecting MB staining, side effects suffered and unusual cases where the dye was invaluable in locating the diseased gland.

Results

A total of 98 patients underwent MB infusion. Of these, 77 cases (78.6%) stained positively with MB and 21 (21.4%) did not. Six patients suffered side effects but there were no cases of neurotoxicity. No positive predictive factors of dye uptake were found. MB was particularly useful in cases of intrathyroidal and ectopic glands as well as improving efficiency in both targeted and open parathyroidectomy.

Conclusions

This series shows that when used correctly, MB is efficacious in locating diseased parathyroid glands, with similar sensitivity rates to preoperative ultrasonography and radionucleotide imaging. Adverse effects were much lower than published previously, which may be attributed to the low dose of MB used (3.5mg/kg).     

红蓝光联合治疗面部中重度痤疮的临床疗效观察

目的:评价红蓝光联合治疗中、重度痤疮的临床疗效。方法:治疗组50例,采用红蓝光联合照射治疗,每位患者每次使用蓝光照射20mi n,然后使用红光照射5～10mi n,每周2次;对照组40例,口服丹参酮胶囊联合外用克林霉素磷酸酯凝胶,两组均治疗4周后判定疗效。结果:红蓝光联合照射治疗中重度痤疮疗效佳,优于对照组(P<0.05)。结论:红蓝光联合照射治疗中、重度痤疮安全有效。