Seminiferous tubule transfection in vitro to define post-meiotic gene regulation

Background  

Post-meiotically expressed genes in the testis are essential for the proper progression of spermatogenesis, and yet, aside from the construction of individual transgenic mice using specific promoters to drive reporter plasmids, there are only very limited possibilities for relevant and quantitative analysis of gene promoters. This is due to the special nature of post-meiotic haploid cells, which to date are not represented in any appropriate cell-lines. This article reports the development of novel methodology using isolated and cultured rat seminiferous tubules in a multiwell format, into which promoter-reporter constructs can be introduced by a combination of microinjection and electroporation.     

Seminiferous tubule degeneration and infertility in mice with sustained activation of WNT/CTNNB1 signaling in sertoli cells

WNT/CTNNB1 signaling is involved in the regulation of multiple embryonic developmental processes, adult tissue homeostasis, abd cell fate determination and differentiation. Many WNTs and components of the WNT/CTNNB1 signaling pathway are expressed in the testis, but their physiological roles in this organ are largely unknown. To elucidate the role(s) of WNT/CTNNB1 signaling in the testis, transgenic Ctnnb1 tm1Mmt/+;Amhr2 tm3(cre)Bhr/+ mice were generated to obtain sustained activation of the WNT/CTNNB1 pathway in both Leydig and Sertoli cells. Male Ctnnb1 tm1Mmt/+;Amhr2 tm3(cre)Bhr/+ mice were sterile because of testicular atrophy starting at 5 wk of age, associated with degeneration of seminiferous tubules and the progressive loss of germ cells. Although Cre activity was expected in Ctnnb1 tm1Mmt/+;Amhr2 tm3(cre)Bhr/+ Leydig cells, no evidence of Cre-mediated recombination of the floxed allele or of WNT/CTNNB1 pathway activation could be obtained, and testosterone levels were comparable to age-matched controls, suggesting that genetic recombination was inefficient in Leydig cells. Conversely, sustained WNT/CTNNB1 pathway activation was obtained in Ctnnb1 tm1Mmt/+;Amhr2 tm3(cre)Bhr/+ Sertoli cells. The latter often exhibited morphological characteristics suggestive of incomplete differentiation that appeared in a manner coincident with germ cell loss, and this was accompanied by an increase in the expression of the immature Sertoli cell marker AMH. In addition, a poorly differentiated, WT1-positive somatic cell population accumulated in multilayered foci near the basement membrane of many seminiferous tubules. Together, these data suggest that the WNT/CTNNB1 pathway regulates Sertoli cell functions critical to their capacity to support spermatogenesis in the postnatal testis.     

Seminiferous tubule fluid and interstitial fluid production. I. Effects of age and hormonal regulation in immature rats

Efferent duct ligation was used to assess seminiferous tubule fluid (TF) production and studies of the kinetics of TF production following this procedure were performed on 25-day-old rats. The rate of TF production was linear for 48 h, thereafter reached a plateau until 72 h and began decreasing at 96 h post-ligation. Using a 16-h ligation period, the onset of TF production was investigated in groups of immature rats from 15 days of age. TF secretion was not detected prior to 15 days but rose rapidly after Day 20 coincident with the prepubertal rise in serum FSH. The acute effect of hormone on TF production following unilateral efferent duct ligation (EDL) was evaluated in 25-day-old rats in which interstitial fluid production (IF) was also assessed in the unligated testis by the method of Sharpe (1977). Single subcutaneous injections of the following hormones were given to groups of rats at the time of EDL: a) NIH follicle-stimulating hormone (FSH) S13 (20 micrograms/rat); b) NIH luteinizing hormone (LH) S22 (200 micrograms/rat); c) testosterone propionate (2 mg/rat); d) human chorionic gonadotropin (hCG) (10 IU/rat); or e) NIH prolactin (Prl) 14 (200 micrograms/rat). A significant rise in TF production occurred following FSH treatment but no effect was noted in any of the other groups. In contrast, a marked stimulation of IF production occurred in rats treated with LH or hCG.     

Seminiferous tubules and daily sperm production in older adult men with varied numbers of Leydig cells

Previous studies of adult men have failed to reveal a relationship between numbers of Leydig cells in the testes and rates of sperm production, perhaps because of a functional excess of these cells in younger men. Hence, a possible relationship between Leydig cell numbers and sperm production was sought in 50 older men, aged 50-90 years, in whom the Leydig cell population had been depleted by age-related attrition. When these men were sorted by increasing numbers of Leydig cells per man into two, three, or five groups, no difference could be found between or within these groups when daily sperm production per man (DSP); seminiferous tubular volume, diameter, or length; or seminiferous epithelial volume was examined. Furthermore, no significant correlation could be detected between Leydig cell numbers and DSP in these 50 men. The only relationship between numbers of Leydig cells and spermatogenesis appeared to be a threshold effect, in that men with fewer than 60 million Leydig cells (4 in this study) had drastically reduced DSP. Men with few Leydig cells tended to have larger Leydig cells, and the increased size was due to more cytoplasm instead of nucleoplasm. There were weak but significant positive correlations between total Leydig cell cytoplasm per man and DSP and between average size of a Leydig cell and DSP. These findings suggest that a relationship may exist between sperm production and the amount of cytoplasm containing testosterone-producing organelles in surviving Leydig cells of older men.     

Prostate cancer in elderly men

Due to increasing life expectancy and the introduction of prostate-specific antigen (PSA) screening, a rising number of elderly men are diagnosed with prostate cancer. Besides PSA serum levels and Gleason score, age is considered to be a key prognostic factor in terms of treatment decisions. In men older than 70 years, treatment without curative intent may deprive the frail patient of years of life. Modern radical prostatectomy techniques are associated with low perioperative morbidity, excellent clinical outcome, and documented long-term disease control. Thus, radical prostatectomy should be considered because local treatment of organ-confined prostate cancer potentially cures disease. The huge extent of PSA screening programs may lead to overdiagnosis of prostate cancer. Not every man who is diagnosed with prostate cancer will develop clinically significant disease. This has led to the concept of expectant management for screen-detected, small-volume, low-grade disease, with the intention of providing therapy for those men with disease progression.     

Intratesticular unconjugated steroids in elderly men

As an extension of our studies on the influence of age on testicular function and with the aim of detecting whether the decline in testosterone production by aged testes is accompanied by a block in the biosynthetic chain leading from cholesterol to testosterone, we determined in the testis of young and elderly men, who died suddenly either from a cardiac incident or from accident, intratesticular steroids: pregnenolone, 17 hydroxypregnenolone (3 beta, 17 alpha-dihydroxy-5-pregnen-20-one), dehydroepiandrosterone, androstenediol, (5-androsten-3 beta, 17 beta-diol), progesterone, 17 hydroxyprogesterone, androstenedione, 17 beta-estradiol as well as testosterone, dihydrotestosterone (5 alpha-androstan-17 beta-ol-3-one) and androstanediol (5 alpha androstane-3 alpha, 17 beta-diol). The intratesticular steroid pattern in elderly men was essentially characterized by a decrease of the 5-ene steroid concentration, whereas we did not observe a decrease in the 4-ene steroids, progesterone concentration being even significantly higher in the aged testes. There was no evidence for a decrease in either lyase or 17-hydroxylase activity. It is suggested that the steroid pattern as observed in the aged testes is the consequence of a decreased oxygen supply, due to a decreased testicular perfusion.     

Compensatory muscle fiber hypertrophy in elderly men.

Muscle strength and muscle morphology have been studied three times during a period of 11 yr in nine elderly men. On the last occasion the average age was 80.4 (range 79-82) yr. Body cell mass decreased by 6% and muscle strength for knee extension, measured by means of isometric and concentric isokinetic (30-60 degrees/s) recordings, declined by 25-35% over the 11-yr period. Between 76 and 80 yr of age only the isokinetic strength for 30 degrees/s decreased significantly. Muscle fiber composition in the vastus lateralis did not change between 69 and 76 yr of age, but there was a significant reduction in the proportion of type IIb fibers from 76 to 80 yr. The decrease in type II fiber areas was not significant between 69 and 76 yr of age (as in a larger sample from the same population), but a significant increase in both type I and type II fiber areas was recorded from 76 to 80 yr of age and biceps brachii showed similar tendencies. In the same period, the enzymatic activities of myokinase and lactate dehydrogenase subsided in the vastus lateralis, but there was no change for triose phosphate dehydrogenase, 3-hydroxy-CoA-dehydrogenase, and citrate synthase. The muscle fiber hypertrophy in this group of elderly men with maintained physical activity between 76 and 80 yr of age is interpreted as a compensatory adaptation for the loss of motor units. In addition, the adaptation with respect to oxidative capacities seems to be maintained at this age.     

'Inadequate' dietary habits and mastication in elderly men

Objective: The aim of this study was to re‐evaluate data about oral status, mastication and nutrition in elderly men in Malmö, Sweden, recorded in 1985–1987, to assess associations between inadequate dietary habits, oral conditions and masticatory function. Materials and methods: Four hundred and eighty‐one men, aged 67–68, participated in a comprehensive health examination, including tooth and denture status and masticatory tests. A separate study of dietary habits and nutritional status was made. Ninety‐five men had inadequate dietary habits. The databases of dental/denture status, mastication, nutritional status and social network factors were re‐evaluated for assessment of associations. Results: No significant differences between those with adequate or inadequate nutrition were found with regard to the number of teeth, occlusal contacts or removable dentures. Also self‐assessed chewing did not show any differences. Conclusion: Inadequate dietary habits were independent of teeth and denture status. Some correlations to social network conditions could be identified. Overweight, obesity, low physical activity and high alcohol intake were more common among those with inadequate nutritional intake.     

Weight stability masks sarcopenia in elderly men and women

Skeletal muscle loss or sarcopenia in aging has been suggested in cross-sectional studies but has not been shown in elderly subjects using appropriate measurement techniques combined with a longitudinal study design. Longitudinal skeletal muscle mass changes after age 60 yr were investigated in independently living, healthy men (n = 24) and women (n = 54; mean age 73 yr) with a mean +/- SD follow-up time of 4.7 +/- 2.3 yr. Measurements included regional skeletal muscle mass, four additional lean components (fat-free body mass, body cell mass, total body water, and bone mineral), and total body fat. Total appendicular skeletal muscle (TSM) mass decreased in men (-0.8 +/- 1.2 kg, P = 0.002), consisting of leg skeletal muscle (LSM) loss (-0.7 +/- 0.8 kg, P = 0.001) and a trend toward loss of arm skeletal muscle (ASM; -0.2 +/- 0.4 kg, P = 0.06). In women, TSM mass decreased (-0.4 +/- 1.2 kg, P = 0.006) and consisted of LSM loss (-0.3 +/- 0.8 kg, P = 0.005) and a tendency for a loss of ASM (-0.1 +/- 0.6 kg, P = 0.20). Multiple regression modeling indicates greater rates of LSM loss in men. Body weight in men at follow-up did not change significantly (-0.5 +/- 3.0 kg, P = 0.44) and fat mass increased (+1.2 +/- 2.4 kg, P = 0.03). Body weight and fat mass in women were nonsignificantly reduced (-0.8 +/- 3.9 kg, P = 0.15 and -0.8 +/- 3.5 kg, P = 0.12). These observations suggest that sarcopenia is a progressive process, particularly in elderly men, and occurs even in healthy independently living older adults who may not manifest weight loss.     

The extrasplanchnic origin of blood dihydrotestosterone in elderly men

The splanchnic extraction and interconversion of testosterone (T) and dihydrotestosterone (DHT) were studied in 5 elderly men undergoing cardiac catheterization using a constant Infusion of [1,2-³H] testosterone and [4-¹⁴C] DHT. Metabolic clearance rate (MCR), splanchnic extraction (SE), splanchnic clearance (SC), extrasplanchnic clearance (ESC), transfer constant In blood ([P]_BB^T-DHT) and transfer constant across the liver ([P]_BB^T-DHT) were calc?ulated. The MCR^T was 675 ± 108 (mean ± SC) L/day and MCR^DHT was 409 ± 68 L/day. SE^T was 45.9 ± 7.0% and SE^DHT was 18.5 ± 5.4%. When these values are compared with those recently reported by us for normal men, there is a $\text{1}\text{3}$ reduction in SE^T and $\text{1}\text{2}$ reduction for SE^DHT in elderly men. The calculated SC^T and ESC^T were 355 ± 72 L/day and 320 ± 86 L/day, respectively. SC^DHT and ESC^DHT were 145 + 48 L/day and 263 ± 77 L/day respectively, suggesting that a major fraction of DHT is metabolized in extrasplanchnic organs. No evidence for a net appearance of DHT by either mass or specific activity analysis in hepatic vein blood was observed indicating that the splanchnic compartment does not contribute DHT into the circulation either by $\text{de novp}$ synthesis or via conversion from testosterone. This work indicates that conversion of testosterone to DHT in elderly men occurs entirely in extrasplanchnic tissue.     

Thermogenic effect of food in physically well-trained elderly men

Basal metabolic rate (BMR) and the thermogenic effect of food (TEF) after a liquid mixed meal of 2092 kJ (500 kcal) were examined in physically well-trained, elderly men in comparison with sedentary weight- and age-matched controls. BMR tended to be higher and TEF was significantly higher in the physically well-trained men than in the controls. No certain differences were found in plasma thyroid hormones or catecholamines. BMR correlated with whole body potassium while TEF did not. The tendency to elevated BMR in the well-trained men might therefore be due to their greater muscle mass. The elevated TEF, however, probably has other causes and might be associated with the elevated catecholamine sensitivity associated with the physically trained condition.     

高龄老年尿钠排泄的特点分析

目的:探讨高龄老年人24 h尿钠排泄特点,为防治老年钠代谢紊乱提供依据.方法:我院老年病房80岁以上老年42例,根据肌酐清除率分为肌酐清除率正常组及异常组.另选中年组24人及老年组31人作为对照.所有患者均检测24 h尿钠、钾、磷、肌酐及肌酐清除率.结果:①高龄组肌酐清除率低于中年组及老年组,24 h尿钠排泄量亦较中年组及老年组减少;高龄组中肌酐清除率异常组的24 h尿钠排泄量较肌酐清除率正常组显著减少.②中年组与老年组尿钠排泄和肌酐清除率无相关关系,高龄组尿钠排泄与肌酐清除有直线相关关系;高龄组肌酐清除率正常组肾脏排钾保钠能力减退、尿钠排泄增加,肌酐清除率异常组钾、钠排泄均减少;③三组的钠摄入量均在6g以上.结论:高龄老年24 h尿钠排泄与肌酐清除率呈直线相关关系.当肾功能处于代偿阶段时,钠排泄增多,但严重肾功能不全时尿钠排泄减少.