氯化镧对雄性小鼠精子质量及睾丸酶活力的影响

探讨氯化镧对小鼠精子质量及睾丸细胞酶的影响。40只成年昆明种雄性小鼠随机分成对照组、低(25mg·kg-1)、中(50mg·kg-1)、高(100mg·kg-1)剂量组,腹腔注射1次/4d,饲养35d。测定睾丸和附睾脏器指数,检测并计算精子数量、活精率、活动率和畸形率以及睾丸碱性磷酸酶(AKP)、酸性磷酸酶(ACP)、乳酸脱氢酶(LDH)、一氧化氮合酶(NOS)活力。结果显示,高剂量氯化镧降低了小鼠睾丸AKP活力,抑制了精子数量和质量;中剂量氯化镧能促进NOS活力,使精子数量减少,对精子质量造成损伤。     

环境雌激素硫丹对根田鼠(Microtus oeconomus)生殖毒性效应

为探讨硫丹的生殖毒性,实验选择健康雄性根田鼠20只,随机分成对照组和实验组,分别注射等剂量生理盐水(6mL/kg)和硫丹溶液(7.0mg/kg)。在染毒的第7天和第14天,实验组和对照组各处死5只根田鼠。通过实验,第1阶段实验组与对照组根田鼠的体重变化不大,睾丸系数差异不明显,精子数、精子活动率下降不明显,精子畸形率明显增加(P0.05)。第2阶段实验组与对照组根田鼠的体重变化不大,睾丸系数差异不明显,精子数、精子活动率下降明显,精子畸形率增加,与第1阶段相比,体重没变化,睾丸系数也无明显变化,精子活动率下降明显(P0.05),精子畸形率增加极显著(P0.01)。因此,环境雌激素硫丹可以引起根田鼠睾丸的精子数下降、精子活动率下降、精子畸形率明显增加。     

环境雌激素硫丹对根田鼠(Microtus oeconomous)生殖毒性效应

为探讨硫丹的生殖毒性.实验选择健康雄性根田鼠20只.随机分成对照组和实验组,分别注射等剂量生理盐水(6 mL/kg)和硫丹溶液(7.0 mg/kg).在染毒的第7天和第14天,实验组和对照组各处死5只根田鼠.通过实验,第1阶段实验组与对照组根田鼠的体重变化不大,睾丸系数差异不明显,精子数、精子活动率下降不明显,精子畸形率明显增加(P<0.05).第2阶段实验组与对照组根田鼠的体重变化不大.睾丸系数差异不明显,精子数、精于活动率下降明显,精子畸形率增加,与第1阶段相比,体重没变化,睾丸系数也无明显变化,精子活动率下降明显(P<0.05),精子畸形率增加极显著(P<0.01).因此,环境雌激素硫丹可以引起根田鼠睾丸的精子数下降、精子活动率下降、精子畸形率明显增加.     

磁处理水对家兔生殖影响的研究

目的:探讨磁处理水对家免睾丸、附睾重量以及精子活力、沃纱、密度的影响。方法:新西兰雄性大白免36只,随机分为实验组和对照组。对照组饮用自来水,实验组饮用磁处理水,磁处理水每天更换,实验进行90天。光镜下检测每只家免附睾尾精子密度,精子活力,精子活率,最后将睾丸、附睾称重。结果:实验组与对照组比较,其睾丸附睾重量明显增加,精子密度增加14 % ,精子活力增加8% ,精子活率增加11% ,两组差异有显著性(P <0 .0 5 )。结论:实验结果提示饮用磁处理水能显著提高家免的生殖能力。     

镉对小鼠精母细胞凋亡及附睾内精子质量的影响

采用人工水质染毒的方法,利用透射电镜技术及流式细胞术（FCM）,探讨重金属镉对小鼠精巢内生殖细胞凋亡及附睾内成熟精子质量的影响.结果表明：各试验组小鼠生殖细胞处于凋亡时期的数量显著高于对照组,凋亡时期的生殖细胞超微结构呈现出线粒体空泡、核膜内陷、染色质周缘化及核固缩等形态特征,表明镉容易引起小鼠生殖细胞凋亡;各试验组精子早期凋亡的比例显著高于对照组,而活性精子的比例显著低于对照组（P<0.05）,其中高剂量组（0.10 mmol·L^-1）精子成活率（75.1%）显著低于对照组和其他试验组,而早期凋亡率（22.6%）则显著高于对照组;高剂量组睾丸生殖细胞DNA断裂率（18.2%）及附睾精子断裂率（26.5%）均显著高于对照组（3.3%、5.6 %）（P<0.05）.各试验组小鼠睾丸内DNA断裂的生殖细胞数量低于附睾内DNA断裂的精子数量.随着添加剂量的增加,小鼠睾丸内生殖细胞及附睾内精子凋亡率逐渐升高.表明小鼠生殖细胞凋亡及DNA损伤数量与镉剂量具有一定相关性.     

双酚A对雄性小鼠生殖毒性的影响

目的观察环境雌激素双酚A（BPA）对雄性小鼠生殖功能的损害作用。方法采用小鼠腹腔注射双酚A,染毒成年小鼠5d,饲养30d。观察小鼠精子畸形率、测定血清中一氧化氮（NO）的含量、一氧化氮合酶（NOS）的活性。以雌二醇（E2）作为阳性对照物。结果BPA染毒组小鼠的精子畸形率较对照组高,睾丸和附睾的脏器系数均低于对照组,血清NO含量、NOS活性均高于对照组。结论一定剂量的BPA对小鼠的精子有致畸作用,并可使血清NO含量、NOS活性增高。     

薄荷油对雄性小鼠的抗生育作用研究

目的探讨薄荷油对雄性小鼠的抗生育作用。方法将60只健康的雄性性成熟昆明小鼠随机分成3个试验组和1个对照组,各试验组小鼠灌胃0.135 g/(kg·d),0.27 g/(kg·d)和0.54 g/(kg·d)的薄荷油0.5%羧甲基纤维素钠(sodium carboxymethylcellulose,CMC)混悬液,对照组灌胃0.5%羧甲基纤维素钠溶液,灌胃1周后按雌雄比2∶1合笼,继续对雄鼠灌胃至19 d,采集雄性小鼠的血液并测定血液生化指标,剖检灌胃雌雄小鼠,统计雌性小鼠的怀孕率,测定雄性小鼠睾丸与附睾的脏器系数及附睾精子活力,并固定睾丸和附睾,研究其组织学变化。结果与对照组相比较,雄性小鼠灌胃薄荷油后,导致雌性小鼠的怀孕率降低,睾丸脏器系数和附睾精子活率极显著降低(P<0.01),精子畸形率极显著升高(P<0.01)。光学显微镜下,试验组小鼠睾丸和附睾均有一定程度的损伤,睾丸间质减少,睾丸各级生精细胞排列疏松,曲细精管内有多核巨噬细胞浸润。附睾具正常的管腔结构,但能明显观察到管腔内精子减少。结论薄荷油对雄性小鼠具有抗生育作用,且主要通过影响精子的生成及破坏生精组织而达到,具有实际应用价值,可进一步开发成环境友好型鼠类抗生育药剂。     

男性不育患者精液生殖细胞微核率的观察

本文研究人类精液中幼稚生殖细胞的微核率和男性不育的关系。在31例病人中,11例为无精症或多精症患者,其精液缺乏幼稚生殖细胞而未能进行微核测定。在其余的20例中,微核率最高可达122‰,最低为16‰。其中9例的微核率与对照组相比有显著升高,高微核率往往还伴随有减数分裂不分离以及出现高比例的二倍体及四倍体精子。还讨论了生殖细胞微核率增高作为不育症的一个可能病因以及微核测定的临床使用价值。     

乙酸铜对雄性小白鼠生殖毒性的影响

以清洁级ICR雄性小白鼠为实验动物,研究不同剂量乙酸铜对小白鼠的生殖毒性。采用小白鼠精子畸形实验及小白鼠骨髓嗜多染红细胞(以下简称PCE)微核实验等方法。分别对成年小白鼠腹腔每天注射0.25~16.00mg/kg8个剂量的乙酸铜,染毒7天。结果表明:乙酸铜对小白鼠的体重增长及睾丸重量具有一定的抑制作用,其中组VI、组VII的最明显。不同剂量的乙酸铜均使雄性小白鼠精子密度(P<0.001)、精子活力明显降低,具有明显的剂量效应。各实验组精子畸形率、PCE微核率均明显高于对照组(P<0.001)(P<0.05或P<0.001),且均随乙酸铜剂量的增加而明显升高。结果表明实验剂量的乙酸铜对ICR雄性小白鼠具有明显的生殖毒性效应。     

白消安致大鼠无精子症模型的建立

目的:利用白消安建立SD(Sprague-Dawley)大鼠少精子症动物模型,为治疗人类男性不育症提供实验依据。方法:本研究取6周龄左右SD大鼠随机分为对照组和实验组,实验组通过腹腔注射不同浓度白消安(15、20、30、40、60 mg/kg体重),每个浓度分单次注射和连续10次累计注射两种方式,注射后每隔10d随机取样检测精子浓度、活力、畸形率以及曲细精管内部结构各项指标,30d出现弱精少精等不育症状后,第40d后停止取样,观察白消安对大鼠精子的作用效果。结果:单次注射15-60mg/kg剂量白消安都会使实验大鼠不同程度死亡,连续10 d累计注射剂量15 mg/kg白消安可破坏大鼠睾丸组织的部分精原干细胞,连续10 d累计注射总剂量20 mg/kg是较为理想的实验浓度,可消除全部精原干细胞和大部分支持细胞,30、40、60 mg/kg可致大鼠死亡或睾丸病变充血。结论:白消安对大鼠生殖细胞有毒害作用,通过腹腔注射一定浓度白消安可建立大鼠少精子症模型,可为外源干细胞体内转分化为生殖细胞提供准确的移植时间和实验依据。     

Reversibility of the reproductive toxicity of gossypol in peripubertal bulls

Gossypol has been shown to impair sperm production in male ruminants. The purpose of this study was to determine if the adverse effects of gossypol on spermatogenesis in peripubertal bulls were reversible. Twenty-eight crossbred Angus bulls were allocated into treated and control groups at 11 months of age. For 8 weeks, treated bulls were fed a ration containing 8 mg of free gossypol per kilogram of body weight per day while control bulls were fed a soybean meal ration free of gossypol. At 28-days intervals, scrotal circumference was measured and semen collected to assess sperm motility and morphology. Seven control and seven treated animals were castrated 56 days after the start of the experiment and the testes were examined histologically. The remaining bulls were fed a gossypol-free diet for 210 days prior to castration. There were significant increases in primary and secondary sperm abnormalities in treated bulls 28 and 56 days after gossypol feeding. The number of sperm with proximal droplets was significantly higher in gossypol-treated bulls, suggesting testicular degeneration. There was no significant effect on the sperm motility, scrotal circumference, or histopathological characteristics of the testes. Four weeks after the end of gossypol feeding, primary and secondary abnormalities were still increased in gossypol-treated bulls, however in subsequent collection periods the percentage of abnormalities were similar between groups. At 210 days, there was no treatment effect on scrotal circumference, and histological characteristics of the testes were not different between groups. The deleterious effects of gossypol on the morphological characteristics of spermatozoa were reversible. Gossypol (8 mg/kg per day for 56 days) increased sperm abnormalities but the effects were reversible.     

Histological description of seminiferous epithelium and cycle length of spermatogenesis in the water shrew Neomys fodiens (Mammalia: Soricidae)

Recently, we examined the spermatogenesis cycle length in two shrews species, Sorex araneus characterized by a very high metabolic rate and a polyandric mating system (sperm competition) resulting in a short cycle and Crocidura russula characterized by a much lower metabolic rate and a monogamous mating system showing a longer cycle. In this study, we investigated the spermatogenesis cycle in Neomys fodiens showing an intermediate metabolic rate. We described the stages of seminiferous epithelium according to the spermatid morphology method and we calculated the cycle length of spermatogenesis using incorporation of 5-bromodeoxyuridine into DNA of the germ cells. Twelve males were injected intraperitoneally with 5-bromodeoxyuridine, and the testes were collected. For cycle length determination, we applied a recently developed statistical method. The calculated cycle length is 8.69 days and the total duration of spermatogenesis based on 4.5 cycles is approximately 39.1 days, intermediate between the duration of spermatogenesis of S. araneus (37.6 days) and C. russula (54.5 days) and therefore congruent with both the metabolic rate hypothesis and the sperm competition hypothesis. Relative testes size of 1.4% of body mass indicates a promiscuous mating system.     

Evaluation of DNA damage in different stages of mouse spermatogenesis after testicular X irradiation

To evaluate whether DNA alterations in mature spermatozoa could stem from DNA damage induced in immature germ cells, testis cells and spermatozoa were analyzed by the comet assay and by the sperm chromatin structure assay 14, 45 and 100 days after in vivo X irradiation of the testes. These times were selected, according to the mouse seminiferous epithelium cycle, to follow the DNA damage induced in different germ cell compartments. The cytotoxic action was assessed by DNA flow cytometric analysis of testicular cells. A dose-dependent increase of DNA damage in testis cells was observed 14 days after irradiation, whereas mature sperm cells were not affected. On the other hand, an increase in DNA strand breaks was seen in spermatozoa 45 days after treatment. DNA damage returned to the control levels 100 days after irradiation. The methods used to evaluate DNA damage gave comparable results, emphasizing the correlation between DNA fragmentation and susceptibility of sperm chromatin to denaturation. Both techniques showed the high radiosensitivity of differentiating spermatogonia. The overall results showed that DNA damage induced in pre-meiotic germ cells is detectable in primary spermatocytes and is still present in mature spermatozoa.     

Expansion of murine spermatogonial stem cells through serial transplantation

Mammalian male germ cells might be generally thought to have infinite proliferative potential based on their life-long production of huge numbers of sperm. However, there has been little substantial evidence that supports this assumption. In the present study, we performed serial transplantation of spermatogonial stem cells to investigate if they expand by self-renewing division following transplantation. The transgenic mouse carrying the Green fluorescent protein gene was used as the donor cell source that facilitated identification and recollection of colonized donor germ cells in the recipient testes. The established colonies of germ cells in the recipient testes were collected and transplanted to new recipients. This serial transplantation of spermatogonial stem cells repopulated the recipient testes, which were successfully performed sequentially up to four times from one recipient to the next. The incubation periods between two sequential transplantations ranged from 55 to 373 days. During these passages, the spermatogonial stem cells showed constant activity to form spermatogenic colonies in the recipient testis. They continued to increase in number for more than a year following transplantation. Colonization efficiency of spermatogonial stem cells was determined to be 4.25% by using Sl/Sl(d) mice as recipients that propagated only undifferentiated type A spermatogonia in their testes. Based on the colonization efficiency, one colony-forming activity was assessed to equate to about 20 spermatogonial stem cells. The spermatogonial stem cells were estimated to expand over 50-fold in 100 days in this experiment.     

Peculiarities of development of mouse male germ cells after intratesticular injection of dipin

It has been shown that a single intratesticular injection of the chemical mutagen dipin (experiment) or saline (control) into mice resulted in significant but reversible morphohistological damage of the spermatogenic epithelium. However, unlike the controls, in mutagenized testes these damages were more pronounced. Thus, the process of restoring a normal pattern of spermatogenesis was slower. In addition, on day 35 of fixation, mature gametes were almost completely absent in the cauda epididymis and a large number of sperm cells with abnormal head shape (58.5 versus 1.7% in the controls) appeared in the testes. Using spermatogonial and meiotic micronucleus assay, we found that dipin did not induce a rise in the number of gross chromosomal mutations in the spermatogonial stem cells (SSCs): on days 35, 56, and 100 postinjection, the incidence of aberrant spermatogonia and round spermatids was not significantly different from the saline control. The degree of gametic chromatin decondensation was evaluated after treatment of the cauda epididymal sperm with sodium dodecyl sulfate (SDS) and dithiothreitol (DTT). Judging by the results of the in vitro sperm chromatin decondensation on days 7, 14, 35, 56, and 100 after the injection of dipin or saline, the number of decondensed nuclei decreased sharply in the studied samples as compared with the sperm from intact animals where sperm cells with fully decondensed chromatin prevailed.     

Clinical aspects of male germ cell apoptosis during testis development and spermatogenesis

Apoptosis appears to have an essential role in the control of germ cell number in testes. During spermatogenesis germ cell deletion has been estimated to result in the loss of up to 75% of the potential number of mature sperm cells. At least three factors seem to determine the onset of apoptosis in male germ cells: (1) lack of hormones, especially gonadotropins and androgens; (2) the specific stage in the spermatogenic cycle; (3) and the developmental stage of the animal. Although male germ cell apoptosis has been well characterized in various animal models, few studies are presently available regarding germ cell apoptosis in the human testis. The first part of this review is focused on germ cell apoptosis in testes of prepubertal boys, with special emphasis on apoptosis in normal and cryptorchid testes. A higher percentage of apoptotic spermatogonia was seen in the cryptorchid testes than in the scrotal testes. The hCG-treatment increased the number of apoptotic spermatogonia. The hCG-treatment-induced apoptosis in spermatogonia had severe long-term consequences in reproductive functions in adulthood. Increased apoptosis after hCG-treatment was associated with subnormal testis volumes, subnormal sperm density and pathologically elevated serum FSH. This finding indicates that increased apoptosis in spermatogonia in prepuberty leads to disruption of testis development. To evaluate the role of apoptosis in human adult testes, apoptosis was induced in seminiferous tubules that were incubated under serum-free conditions in the absence or presence of testosterone. Most frequently apoptosis was identified in spermatocytes. Occasionally some spermatids also showed signs of apoptosis. In short term incubations apoptosis was suppressed by testosterone. Our findings lead to the conclusion that apoptosis is a normal, hormonally controlled phenomenon in the human testis. The role of apoptosis in disorders of spermatogenesis remains to be established.     

L-carnitine counteracts prepubertal exposure to cisplatin induced impaired sperm in adult rats by preventing germ cell apoptosis

We investigated the possible protective effects of L-carnitine on cisplatin induced prepubertal gonadotoxicity and on adult sperm. Prepubertal 30-day-old male rats were divided randomly into three groups: control (n = 12), cisplatin exposed (n = 16) and carnitine treated after cisplatin exposure (n = 16). Rats in the experimental groups were injected with a single dose of cisplatin. L-carnitine was injected 1 h before cisplatin administration and for the following 3 days for the cisplatin + carnitine group. The rats were sacrificed at 31 or 90 days old and their testes were harvested for morphometric and histopathological analysis. Testes of 31-day-old prepubertal rats were examined for germ cell apoptosis using the TUNEL method and for proliferation using PCNA immunostaining. The morphology, motility, quantity and vitality of sperm in epididymal fluid samples of adult 90-day-old rats also were evaluated. L-carnitine treatment reduced testicular damage and the number of TUNEL positive cells significantly, while the number of PCNA positive cells in the cisplatin + carnitine group increased compared to the cisplatin group. During the adult period, epididymal sperm count and viability were improved in rats treated with L-carnitine before prepubertal cisplatin injection. L-carnitine may reduce late testicular and spermatic damage caused by cisplatin administration to prepubertal rats by inducing germ cell proliferation and preventing apoptosis.     

Ubiquitin C-terminal hydrolase L-1 is essential for the early apoptotic wave of germinal cells and for sperm quality control during spermatogenesis

Ubiquitination is required throughout all developmental stages of mammalian spermatogenesis. Ubiquitin C-terminal hydrolase (UCH) L1 is thought to associate with monoubiquitin to control ubiquitin levels. Previously, we found that UCHL1-deficient testes of gad mice have reduced ubiquitin levels and are resistant to cryptorchid stress-related injury. Here, we analyzed the function of UCHL1 during the first round of spermatogenesis and during sperm maturation, both of which are known to require ubiquitin-mediated proteolysis. Testicular germ cells in the immature testes of gad mice were resistant to the early apoptotic wave that occurs during the first round of spermatogenesis. TUNEL staining and cell quantitation demonstrated decreased germ cell apoptosis and increased numbers of premeiotic germ cells in gad mice between Postnatal Days 7 and 14. Expression of the apoptotic proteins TRP53, Bax, and caspase-3 was also significantly lower in the immature testes of gad mice. In adult gad mice, cauda epididymidis weight, sperm number in the epididymis, and sperm motility were reduced. Moreover, the number of defective spermatozoa was significantly increased; however, complete infertility was not detected. These data indicate that UCHL1 is required for normal spermatogenesis and sperm quality control and demonstrate the importance of UCHL1-dependent apoptosis in spermatogonial cell and sperm maturation.     

Effects of acid sphingomyelinase deficiency on male germ cell development and programmed cell death

Deficiency of acid sphingomyelinase (ASM), an enzyme responsible for producing a pro-apoptotic second messenger ceramide, has previously been shown to promote the survival of fetal mouse oocytes in vivo and to protect oocytes from chemotherapy-induced apoptosis in vitro. Here we investigated the effects of ASM deficiency on testicular germ cell development and on the ability of germ cells to undergo apoptosis. At the age of 20 weeks, ASM knock-out (ASMKO) sperm concentrations were comparable with wild-type (WT) sperm concentrations, whereas sperm motility was seriously affected. ASMKO testes contained significantly elevated levels of sphingomyelin at the age of 8 weeks as detected by high-performance, thin-layer chromatography. Electron microscopy revealed that the testes started to accumulate pathological vesicles in Sertoli cells and in the interstitium at the age of 21 days. Irradiation of WT and ASMKO mice did not elevate intratesticular ceramide levels at 16 h after irradiation. In situ end labeling of apoptotic cells also showed a similar degree of cell death in both groups. After a 21-day recovery period, the numbers of primary spermatocytes and spermatogonia at G2 as well as spermatids were essentially the same in the WT and ASMKO testes, as detected by flow cytometry. In serum-free cultures both ASMKO and WT germ cells showed a significant increase in the level of ceramide, as well as massive apoptosis. In conclusion, ASM is required for maintenance of normal sphingomyelin levels in the testis and for normal sperm motility, but not for testicular ceramide production or for the ability of the germ cells to undergo apoptosis.     

A comparative morphological study of human germ cells in vitro or in situ within seminiferous tubules.

For many infertile couples, intracytoplasmic germ cell/spermatozoon injection into unfertilized eggs may be their only hope for producing their own biological children. Thus far, success with injection of pre-spermatozoan germ cells such as round spermatids has not been as great as that of spermatozoon injection. This could be due in part to the difficulty of identifying younger (less mature) male germ cells in testicular biopsy dispersions. To improve the identification of various types of live, dispersed, human testicular cells in vitro, a comparative study of the morphological characteristics of human spermatogenic germ cells in vitro or in situ within seminiferous tubules was conducted. Live human testicular tissue was obtained from an organ-donating, brain-dead person with a high density of various germ cells. A cell suspension was obtained by enzymatic digestion, and cells were cultured for 3 days in an excessive volume (100-fold medium:cells; v:v) of HEPES-TC 199 medium at 5 degrees C and observed live with Nomarski optics (interference-contrast microscopy). For comparative purposes, testes from ten men obtained at autopsy were fixed, embedded in epoxy resin, sectioned at 20 microm, and observed unstained by Nomarski optics. This approach allowed comparison of morphological characteristics of individual germ cells seen in vitro or in situ in the human testis. In both live and fixed preparations from control men with varied daily sperm production rates, Sertoli cells have oval to pear-shaped nuclei with indented nuclear envelopes and large nucleoli, which makes their appearance distinctly different from germ cells. The size, shape, and chromatin pattern of nuclei, and the presence of meiotic metaphase figures, acrosomic vesicles/structures, tails, and/or mitochondria in the middle piece of germ cells are characteristically seen in live cells in vitro and in those cells observed in the fixed seminiferous tubules. Hence, this comparative approach allows verification of the identity of individual germ cells seen in vitro and provides a checklist of distinguishing characteristics of live human germ cells, to be used by scientists and technical staff in infertility clinics when selecting specific germ cells from a testicular aspirate or enzymatically digested biopsy.