Postnatal testicular development and actin appearance in the seminiferous epithelium of the Habu,Trimeresurus flavoviridis

The postnatal testicular development and actin distribution in the seminiferous epithelium were examined by light microscopy, using the testes of the Habu (Trimeresurus flavoviridis; snake) from 0-year-old to 3-year-old. At 0-year-old (about 1 month after birth), the testis was quite small in size, and the seminiferous epithelium was composed of only Sertoli cells and large spermatogonia. Actin immunoreactivity was observed in the peritubular myoid cells, but could not be detected in the seminiferous epithelium. At 1-year-old (about 10 months after birth), the testicular size increased to a great degree. In the seminiferous epithelium, spermatocytes newly appeared. Actin could still not be detected in the seminiferous epithelium. At 2-year-old (about 1 year and 10 months after birth), the testes continued to develop in size. In the seminiferous epithelium, elongate spermatids and round spermatids were frequently seen, in addition to Sertoli cells, spermatogonia and spermatocytes. Thus, active spermatogenesis was clearly recognized at this age. Moreover, the actin distribution in the seminiferous epithelium was observed at the site between Sertoli cells and spermatids, as well as that at adult stage. The immunoreactivity of actin in the peritubular myoid cells gradually increased from 0-year-old to 2-year-old. Conclusively, it seems likely that spermatogenesis in the Habu initiates at 2-year-old, accompanying with the appearance of actin in the seminiferous epithelium.     

鸡睾丸生精上皮细胞冷冻保存研究

以广西土鸡为试验材料，利用两步酶解法从出壳后20～30日龄公鸡睾丸中获取生精上皮细胞，以含20％胎牛血清的DMEM为基础培养液，比较了不同浓度DMSO和蔗糖对生精上皮细胞冷冻保存效果的影响。结果表明：DMSO的最佳浓度为10％，添加蔗糖对冻存有害。以10％DMSO为防冻剂，复苏后生精上皮细胞原代混合培养可形成典型的精原干细胞集落，集落呈碱性磷酸酶（AKP）阳性，说明以10％DMSO为防冻剂的冷冻体系适合鸡睾丸生精上皮细胞的冷冻保存。     

The cycle of the seminiferous epithelium in the greater Japanese shrew mole, Urotrichus talpoides

Spermatogenesis and acrosomal formation in the greater Japanese shrew mole, Urotrichus talpoides, were studied by light microscopy. On the basis of acrosomal changes, morphology of spermatid head, nuclear shape, appearance of meiotic figures, location of spermatid and period of spermiation, the cycle of the seminiferous epithelium was classified into 12 stages, and developing spermatids could be divided into 15 steps. The mean relative frequencies of stages from I to XII were 10.9, 8.7, 9.8, 7.3, 8.5, 10.3, 12.5, 8.7, 5.8, 5.4, 5.1 and 7.1%, respectively. Similar to the case in the musk shrew, the spermatid nucleus of the greater Japanese shrew mole remained in the middle region of the seminiferous epithelium and only the acrosome extended towards the basement membrane. The elongation of the acrosome, however, was not prominent. The proacrosomal vesicle first appeared in stage II and then one large and round granule was seen in stage III. The acrosomal vesicle became flattened on the surface of the nucleus in stage IV. Spreading of the acrosomic system has been recognized from stage VII. In stage VII, spermiation occurred. In stage IX, the spermatid nucleus began to elongate. Elongation and condensation of the nucleus were clearly observed in stage X. In stage XII, pachytene spermatocytes divided into diplotene spermatocytes. In stage XII, meiotic figures and secondary spermatocytes were observed.     

Morphological features of the seminiferous epithelium in cat (Felis catus, L. 1758) testes

The aim of the present study was to assess the changes in the germinal epithelium in cats of different ages. Routine histological staining was applied to perform morphological and stereological examinations. The animals were divided into five groups according to age: under 8 months (n=28), 8-12 months (n=30), 12-36 months (n=33), 3-6 years (n=14) and older than 6 years (n=13). The appearance of the gonads of the males in the first group varied the most. The seminiferous tubules of the youngest cats consisted of a monolayer of supporting cells and a few spermatogonia. No tubular lumina were present, and the diameters of the seminiferous tubules reached 132.5 microm. We noted the typical arrangement of gametogenic cells with a tubule diameter of 191.83 microm in the second group. We observed multilayer germinal epithelia with the most significant production of gametes and a seminiferous tubule diameter of 202.61 microm in the third group. The diameters of the seminiferous tubules of the forth and fifth groups were 193.38 microm and 191.84 microm, respectively. The obtained data revealed that the most intensive morphological diversification of the seminiferous epithelium in cats occurs at about 7-8 months of age. The diameters of seminiferous tubules were highest in the third group of cats, and the activity of spermatogenesis of this group, expressed as the number of sperm per 10 mm(2), was also the most distinctive. The spermatogenesis process was most evident in cats between 12 and 36 months of age, which was also when the sperm concentration was highest per estimated surface.     

Spontaneous vacuolar degeneration of the thyroid follicular epithelium in cynomolgus monkeys

Vacuolar degeneration of the thyroid follicular epithelium was observed in two untreated female cynomolgus monkeys assigned to control groups. In light microscopy, large vacuoles containing a homogenous substance occupied the basal region of the epithelium, and the nuclei had shifted toward the apical region. The vacuoles showed negative reactions to PAS and thyroglobulin. Electron microscopic observation revealed dilatation of the rough endoplasmic reticulum corresponding to the vacuoles. The plasma TSH, T3 and T4 levels determined for the samples kept frozen were within the normal ranges, suggesting that the thyroid function was kept intact.     

Localization of smooth muscle actin in the iridocorneal angle of normal and spontaneous glaucomatous beagle dogs

Purpose To date, our knowledge of the canine trabecular meshwork (TM) with regard to contractility is incomplete. It is important to understand the potential contractile capability within the TM and possible changes associated with spontaneous hypertensive glaucoma. To that end we have examined the presence of actin, including smooth muscle (SM) actin, in the normal and glaucomatous canine iridocorneal angle (ICA) morphologically and immunohistochemically. Methods Sections from the ICAs of 12 Beagles with inherited glaucoma (3 months to 6 years old) and age‐matched normal Beagles were treated with target retrieval, protein and power blocked and sequentially incubated with the primary antibody (rat anticanine SM actin) and the secondary antibody (rabbit antirat immunoglobulin), followed by peroxidase labeled streptavidin and incubation with substrate‐chromogen solution (AEC). Smooth muscle fibers that lined an artery within canine heart tissue were used as positive controls. Separate specimens were prepared for ultrastructual observation. Results Ultrastructurally, cells within the inner, posterior region of the corneoscleral TM and outer, posterior region of the uveal TM contained many microfilaments, 6 nm in diameter (i.e. actin). Immunohistochemistry demonstrated that cells within these regions possessed SM actin, having been greatest posteriorly, but extended anteriorly to a lesser extent. In the preglaucomatous affected dog the localization pattern for SM actin was identical to that seen in the normal dogs. With the progression of the disease the pattern disappeared. Conclusions The interior presence of myofibroblastic cells within the canine ICA suggests that these cells and the smooth muscle cells of the ciliary body along the same plane of orientation function to facilitate the removal of aqueous humor and are likely to be influenced by vascular mediators. The contractile apparatus for the ICA in the dog with inherited glaucoma appeared identical to that of the normal dog prior to expression of the disease, but weakened as the disease progressed.     

C6, a new monoclonal antibody,reacts with the follicle-associated epithelium of calf ileal Peyer's patches

The follicle-associated epithelium (FAE) of Peyer''s patches (PPs) contains M cells that are important for reducing mucosal immune responses by transporting antigens into the underlying lymphoid tissue. We generated a monoclonal antibody (C6) that reacted with the FAE of calf ileal PPs, and analyzed the characteristics of C6 using immunohistochemistry and Western blotting. FAE of the ileal PP was stained with C6 during both late fetal developmental and postnatal stages. Neither the villous epithelial cell nor intestinal crypt basal cells were stained at any developmental stage. During the prenatal stages, FAE of the jejunal PP was C6-negative. However, a few C6-positive cells were distributed diffusely in some FAE of the jejunal PPs during the postnatal stages. The protein molecular weight of the antigen recognized by C6 was approximately 45 kDa. These data show that C6 is useful for identifying the FAE in ileal PPs and further suggest that differentiation of the FAE in these areas is independent of external antigens.     

Potential of triptolide in reproductive management of the house rat,Rattus rattus

Mature and healthy male house rats, Rattus rattus (n= 160) were fed on bait (cracked wheat: powdered sugar, 98:2) containing different concentrations of triptolide (0.1, 0.05, 0.025 and 0%) for 7 and 14 days in no‐choice and bi‐choice feeding tests in the laboratory. The objective of the study was to record the antifertility affects of triptolide after 30 and 60 days of termination of treatment. Results revealed no significant effect of triptolide treatment on weights of testis, epididymis, seminal vesicles and prostate gland of rats. Overall, sperm motility, live sperm count, sperm density and sperm morphology in the cauda epididymal fluid were found to differ significantly (P≤ 0.05) between untreated and treated groups of rats. The major effect of triptolide on sperm morphology was in the form of sperm head tail separation, which was up to 56.0% in rats treated for 14 days in no‐choice and autopsied after 30 days. A significant effect (P≤ 0.05) of triptolide treatment was observed on the histomorphology of the testis, which included a dose‐dependent decrease in diameter of seminiferous tubules, thickness of germinal epithelium and numbers of various spermatogenic cells. Cell associations in the seminiferous epithelial cycle were poorly developed in rats ingesting medium (4.7–5.1 mg/100 g bw) and high doses (6.9–7.2 mg/100 g bw) of triptolide than rats ingesting low doses (1.8–2.3 mg/100 g bw) and untreated rats. The cell stages affected had not recovered fully within the 60 day period following triptolide withdrawal. The present study suggests the potential of triptolide in reproductive management of Rattus rattus.     

Increases in the expression of Na+/H+ exchanger 1 and 3 are associated with insulin signalling in the ruminal epithelium

Na⁺/H⁺ exchanger (NHE), which catalyses the exchange of extracellular Na⁺ for intracellular H⁺, is of importance in the maintenance of Na⁺ and pH homoeostasis for rumen epithelial cells. Studies in ruminants showed that high concentrate diets could increase the expression of NHE in ruminal epithelium. Results of recent studies further indicated that insulin, as an important hormone closely related to dietary concentrate, could enhance the expression of NHE. In this study, we have investigated the mechanisms of insulin regulating the expression of NHE in rumen epithelial cells and its potential role in dietary modulation of NHE expression in ruminal epithelium of cows. In primary culture, insulin increased phosphorylation of ERK 1/2 and AKT in rumen epithelial cells. However, this promotion was diminished by insulin receptor inhibitor. Insulin also stimulated NHE1 and NHE3 expression. But this increase was suppressed by insulin receptor inhibitor, ERK inhibitor and AKT inhibitor. In the present animal experiment, NHE1 and NHE3 expression increased in rumen epithelium of cows ingesting a high concentrate diet (HC, 60% concentrate), accompanied by increased insulin concentration in plasma, compared to those feeding a low concentrate diet (LC, 20% concentrate). Furthermore, the phosphorylation of ERK1/2 and AKT was higher in the rumen epithelium of the HC group than those in the LC group. Collectively, these results indicate that diet‐dependent change of NHE1 and NHE3 abundance was mediated, at least in part, by plasma insulin through the ERK and AKT pathway.     

Testicular histomorphometry and the proliferative and apoptotic activities of the seminiferous epithelium in Syrian hamster during spontaneous recrudescence after exposure to short photoperiod

Syrian hamsters are photoperiodic rodents in which reproduction, including testicular function, is stimulated by long photoperiod exposure and curtailed by exposure to a short photoperiod. The objectives of this study were to characterize the testis histomorphometrically and to determine the role of the proliferation and apoptosis phenomena in the recovery of the seminiferous epithelium during spontaneous recrudescence after exposure to short photoperiod. The study was performed using conventional light microscopy, proliferating cell nuclear antigen and terminal deoxynucleotidyl transferase (TdT)‐mediated dUTP in situ nick end labelling staining, image analysis software, and transmission electron microscopy in three recrudescence groups: initial recrudescence (IR), advanced recrudescence (AR) and total recrudescence (TR). The results morphometrically pointed to the gradual recovery of the testicular and tubular volumes, as well as of the seminiferous epithelium. Among the IR and AR groups, the increase in testicular and tubular volumes was accompanied by an increase in tubular diameter and length, with an increase in interstitial volume. From AR to TR, there was an increase in the tubular and total volumes, but, in this case, with a gradual increase in tubular diameter. Recovery of the seminiferous epithelium was accompanied by changes in apoptosis and proliferation activities. The first decreased halfway through the process, and the second remained higher than the control levels throughout the recrudescence stage. Alterations in the spermatozoa were ultrastructurally observed, which indicated that spermiogenesis was not yet completely normal. In conclusion, spontaneous testicular recrudescence in Syrian hamster comprises two histomorphometrical phases: the first related to an increase in tubular length and diameter and interstitial volume and the second depending principally on the gradual increase in tubular diameter. The restoration of the seminiferous epithelium is due to apoptosis reaching normal values in the AR group accompanied by higher proliferative activity than that observed in the Control group.     

黄体生成素和褪黑素受体在不同日龄雏鸵鸟睾丸内的分布研究

采用免疫组织化学SABC法对1 d、30 d和90 d雏鸵鸟睾丸内黄体生成素(1uteinizing hormone,LH)和褪黑素受体(melatonin receptor,MR)的分布进行定位,结果显示:LH阳性细胞胞质或胞膜出现棕黄色,胞核无阳性反应;MR阳性反应颗粒呈棕褐色或棕黄色,位于胞质、胞膜或胞核,呈散在...     

高谷物日粮促进山羊瘤胃上皮单羧酸转运蛋白1及钠钾ATP酶mRNA的表达

本试验旨在研究高谷物日粮对山羊瘤胃上皮形态结构及单羧酸转运蛋白(monocarboxylate transporter, MCT)和钠钾ATP酶mRNA表达的影响。将10头装有永久性瘤胃瘘管的健康阉割公山羊随机分为饲喂全粗料日粮的对照组(Hay,0%谷物,n=5)和饲喂高谷物日粮的处理组(HG,65%谷物,n=5),试验期为7周。试验开始后,于每周晨饲后的0、2、3、4、6、8和12 h连续采集瘤胃液监测瘤胃pH值的变化,收集其中第0、3、6和12 h的瘤胃液待测挥发性脂肪酸(volatile fatty acid, VFA)浓度。试验的第50天,屠宰采集瘤胃上皮用于形态学及基因定量分析。研究结果显示:与全粗料组山羊相比,高谷物组山羊瘤胃pH值、乙酸浓度及乙丙比都显著下降(P<0.001),而瘤胃丙酸浓度、丁酸浓度及其他VFA浓度都显著升高(P<0.001);高谷物日粮组的瘤胃乳头长度显著高于对照组(P=0.001),瘤胃乳头宽度显著低于对照组(P<0.001),但是两组间的瘤胃乳头表面积并无显著差异;透射电镜结果显示,长期饲喂高谷物日粮导致瘤胃上皮细胞线粒体发生降解;实时定量PCR结果表明,与对照组相比,高谷物日粮显著升高了MCT1(P<0.001)和钠钾ATP酶(P=0.001)的mRNA表达量,显著降低了MCT4的mRNA表达量(P=0.041),但对MCT2的表达没有显著影响(P=0.305);进一步分析这些基因的mRNA表达量与pH值和VFA浓度之间的相关性,结果显示,MCT1和钠钾ATP酶的mRNA表达量与瘤胃pH值和乙酸浓度呈显著负相关,与总VFA、丙酸、丁酸的含量呈显著正相关,而MCT4的mRNA表达量与pH值呈显著正相关,与总VFA、丙酸、丁酸的含量呈显著负相关。以上结果提示:高精料引起的瘤胃pH值下降和VFA的变化可能与瘤胃上皮MCT和钠钾ATP酶表达量的变化相关。研究结果对深入认识高谷物饲喂引发的瘤胃功能紊乱具有重要意义。     

用原子荧光法检测家蚕体内硒的含量及其分布

硒对动物生殖发育、免疫系统具有重要功能,是动物必需微量元素。采用原子荧光法对不同系统家蚕体内硒的含量及其分布进行测定,结果显示:在喂食相同桑叶的情况下,不同系统家蚕品种幼虫体内的硒含量存在显著差异,5龄第4天的中系家蚕品种幼虫体内的硒含量明显高于日系家蚕品种,其中中系家蚕品种19-570体内的硒含量最高。对家蚕品种大造体内硒分布的研究结果表明:精巢中的硒含量明显高于其他组织,头部次之,脂肪体中的硒含量相对较低,由此推测硒在家蚕的生殖发育过程中可能发挥着重要作用。     

Histomorphometrical study of the testis in the desert Rodent,Gerbillus tarabuli,sexually immature and adult during the annual reproductive cycle

Our interest in the testicular study of the desert rodent, Gerbillus tarabuli, would provide a better comprehension of the seasonality of spermatogenesis in rodents. We thus performed a biometric, histomorphometric and statistical analyses in Gerbillus tarabuli sexually immature and adult during the annual reproductive cycle (active and regressed states of gonadal activity). For that, fixed testes were embedded in paraffin. Sections were stained using the modified Heidenhain Azan and then were subjected to morphometric measurements at the light microscopic level. The biometric study revealed a positive correlation between the body weight and the maturity state only, with an average of 20.8 ± 2.92 g in sexually immature Gerbils, 37.40 ± 1.86 g in the sexually quiescent adults (p < .01) and 40.40 ± 2.98 g in the sexually active adults (p > .05), while the testes weight (sexually immatures = 0.02 ± 0.003 g, sexually quiescent adults = 0.13 ± 0.01 g; p < .001 and sexually active adults = 0.27 ± 0.01 g; p < .001) and the gonadosomatic index (sexually immatures = 0.09 ± 0.02%, sexually quiescent adults = 0.36 ± 0.04%; p < .01 and sexually active adults = 0.68 ± 0.07%; p < .01) varied according to the maturity state and as well as to the season in the adults. Through the histomorphometric study, the tubular diameter showed a continuous increase from sexually immature Gerbils (72.83 ± 7.57 μm) to sexually quiescent adults (99.45 ± 4.34 μm) p < .05 then to sexually active adults (182.58 ± 5.22 μm); p < .001, this is explained by the appearance of a large lumen after puberty in the quiescent (26.32 ± 1.28 μm); p < .001 and the enlargement of the seminiferous epithelium in actives (75.11 ± 1.81 μm); p < .001. Besides, the nucleo-cytoplasmic ratio of Leydig cells (sexually immatures = 0.5 ± 0.02) diminishes significantly after puberty in the rest period (0.42 ± 0.03); p < .05 and then during the active period (0.24 ± 0.01); p < .001. These striking biometric and morphometric differences in Gerbillus tarabuli testis between sexually immature Gerbils, sexually quiescent and active adults, follow the photoperiod-induced changes in endocrine and exocrine functions and prove that Gerbillus tarabuli is a useful model to study histo-physiological relationships in the testis in order to develop a more complete understanding of spermatogenesis phenomenon.     

Presence of opioid growth factor and its receptor in the normal dog,cat and horse cornea

Objective To determine if opioid growth factor (OGF, [Met⁵]enkephalin) and its specific receptor (OGFr) are present in normal cat, dog and horse cornea. Animals studied Normal dog, cat and horse. Procedure Corneas were obtained from animals euthanized for reasons unrelated to this project. One cornea from each of three normal cats, dogs and horses was evaluated. The right or left cornea from each animal was chosen randomly. Corneas were harvested and placed in corneal storage media for transport to The M.S. Hershey Medical Center of The Pennsylvania State University where immunocytochemistry techniques were used to demonstrate the presence and location of OGF and OGFr. Tissues were rinsed in Sorenson's phosphate buffer, immersed in 20% sucrose in buffer and then snap frozen in isopentane. Corneas were then embedded in OCT medium and 15 µm cryostat sections were created. Presence of OGF was determined by using a polyclonal antibody to [Met⁵]enkephalin and assessing immunoreactivity. OGFr presence was determined by using a previously characterized rabbit polyclonal antibody to the receptor. Results OGF and OGFr were identified in large quantities in the corneal epithelium of all three species. Conclusion Opioid growth factor and its specific receptor are present in the corneal epithelium of normal cats, dogs and horses. OGF is present in the cornea of many species and its presence is theorized to inhibit healing of injured tissue.     

Changes in testicular size,echotexture, and arterial blood flow associated with the attainment of puberty in Dorper rams raised in a subtropical climate

There is a paucity of information on the relationships of testicular morphology, echotextural attributes, and blood flow dynamics with pubertal development of rams raised in a subtropical climate. Forty‐five Dorper rams (24 rams aged 8–11 months and 21 rams aged 12–24 months) were examined using a portable ultrasound scanner connected to a 7.5‐MHz transducer. Computer‐assisted analyses of testicular ultrasonograms utilized commercially available Image ProPlus^® analytical software. Spectral Doppler scans of testicular arteries were performed immediately after scrotal (B‐mode) ultrasonography to determine peak systolic velocity (PSV), end‐diastolic velocity (EDV), resistive index (RI = [PSV?EDV]/PSV), and pulsatility index (PI = [SPV–EDV]/mean velocity) of the blood vessels. The length of the testes (9.7 ± 0.3 compared with 9.0 ± 0.2 cm) and scrotal circumference (33.3 ± 0.5 compared with 31.8 ± 0.4 cm) were greater (p < 0.05) but testicular depth (4.5 ± 0.1  compared with 4.9 ± 0.08 cm) was less (p < 0.05) in sexually mature compared with peripubertal rams. [Corrections added on 9 Jan 2019 after initial online publication: The testicular size values in the sentence were corrected.] There were no differences (p > 0.05) between the two age groups of Dorper rams in blood flow indices of testicular arteries. Mean numerical pixel values (100.5 ± 4.1 compared with 89.2 ± 4.8) and pixel heterogeneity (25.6 ± 0.6 compared with 23.6 ± 0.5) of testicular parenchyma were greater (p < 0.05) in peripubertal than in postpubertal rams. Semen volume was negatively correlated with PI of testicular arteries (r = ?0.57, p = 0.04). In summary, the attainment of sexual maturity in the rams of the present study was associated with significant changes in testicular length and depth, scrotal circumference, and parenchymal echogenicity/hetrogeneity but not in testicular volume and blood perfusion rates. Testicular artery PI can be used to predict the volume of ejaculate in rams.     

AR、ER基因在鸡、鹌鹑及其杂交种睾丸组织中的表达及其对睾丸生长发育的影响

为探讨鸡（♂）与鹌鹑（♀）属间杂交种睾丸生长发育不良的分子机理,采集新罗曼蛋用型公鸡、朝鲜公鹌鹑、鸡与鹌鹑杂交的雄性杂交种不同发育时期的睾丸组织样共计96份并测定其体质量与睾丸质量。采用实时荧光定量PCR对公鸡、公鹌鹑及其杂交种不同生长发育期睾丸组织中AR、ER基因的mRNA表达进行了检测。结果显示：（1）鸡和鹌鹑的睾丸均能随着日龄、体质量的增长而正常生长发育,但鸡与鹌鹑杂交的杂交种的睾丸却生长缓慢而发育不良;（2）鸡和鹌鹑不同生长发育期睾丸组织中AR、ER基因mRNA的表达具有相似性,AR基因均有一个极显著的峰值,ER基因均呈波动性变化;鸡与鹌鹑的杂交种均为极端性表达,AR基因呈直线下降,ER相反则一直升高;与鸡和鹌鹑相比,杂交种AR、ER基因mRNA的极端性表达均应为异常表达。（3）鸡、鹌鹑及其杂交种的共同点是AR表达为最高峰时,ER则趋于最低点,反之,ER表达为最高峰时,AR则趋于最低点,表明ER基因对AR基因具有上调作用或者AR基因对ER基因具有下调作用,从而推测AR与ER即相对立而又协同作用的表达模式是鸡和鹌鹑睾丸组织正常生长发育的分子调控模式,探明了鸡与鹌鹑的杂交种睾丸组织发育不良的分子影响因素是其睾丸组织中的AR、ER基因mRNA异常表达。