Stereological and morphological analysis of the effects of aging on spermatogenesis in rat testis

Abstract Purpose: To quantify, through stereological and morphometric analysis, spermatogenesis in rats undergoing the natural aging process. Methods: Seventy-two male Wistar rats were divided into 6 equal groups according to age at the time of killing: 3, 6, 9, 12, 18, and 24 months. All the rats were subjected orchiectomy and collection of testicular parenchymal fragments for histological and morphometric analysis. The numerical density of spermatids was calculated using a stereological study, and morphometric analysis was conducted to measure the height of the germinal epithelium and the area of the seminiferous tubules. Results: We found that the 18 and 24 months groups showed a significant reduction in the number of round spermatids. However, the height of the germinal epithelium was not significantly different between the groups. The area of seminiferous tubules was also significantly reduced in the elderly rats compared to that in the young ones. Conclusion: Aging of rats showed a significant reduction in the number of round spermatids and the area of the seminiferous tubules, more pronounced in the rats at 18 and 24 months of life.

Application of flow cytometry in assisted reproductive technology / 中华检验医学杂志

Flow cytometry (FCM) is used for multi parameter and rapid quantitative analysis of biological particles,such as all cells,microorganisms and synthetic microspheres in fast line flow state.It is also a modern cell analysis technology for the separation of specific groups.In recent years,FCM has been applied in the field of assisted reproductive medicine.FCM plays an important role in the diagnosis of immune infertility and predicting the fertilization ability of sperm.This article aims to review FCM application in peripheral immune cell surface marker detection for infertility patients,and research on the structure and function of sperm cell.

Acridine orange staining for fast identification ofthe spermatogenic cells during STA-PUT velocity sedimentation / 基础医学与临床

Objective To establish a fast and accurate technique of identifying the pachytene spermatocytes,round spermatids and elongating and condensing spermatids during STA-PUT velocity sedimentation.Methods Using STA-PUT velocity sedimentation method to isolate the pachytene spermatocytes, round spermatids and elongating and condensing spermatids from mouse testes.To determine the cell populations` distribution,each tube of cell fraction was then partially transfered to the 96 plate well,and each well was added with acridine orange dye.Then each well was analyzed using fluorescence microscopy.Results Three types of spermatogenic cells can be identified quickly and accurately by it`s specific cytoplasm/nucleus character using the acridine orange dye staining under fluorescence detection.Conclusions A rubust method to quickly and accurately determine the pachytene spermatocytes,round spermatids and elongating and condensing spermatids during STA-PUT velocity sedimentation is successfully developed.

Immunohistochemical localization of glucose transporter 1 and 3 in the scrotal and abdominal testes of a dog / 한국실험동물학회지

Glucose is essential for testicular function; the uptake of carbohydrate-derived glucose by cells is mediated by glucose transporters (GLUTs). In the present study, we investigated the activity of GLUT1 and GLUT3, the two main isoforms of GLUTs found in testes, in the left scrotal and right abdominal testes of a German Shepherd dog. Immunohistochemical analysis showed that GLUT1 immunoreactivity was absent in the scrotal and abdominal testes. In contrast, weak to moderate GLUT3 immunoreactivity was observed in mature spermatocytes as well as spermatids in the scrotal testis. In the abdominal testis, relatively strong GLUT3 immunoreactivity was detected in Leydig cells only and was absent in mature spermatocytes and spermatids. GLUT3 immunoreactivity was significantly decreased in the tubular region of abdominal testis and significantly increased in the extra-tubular (interstitial) region of abdominal testis compared to observations in the each region of scrotal testis, respectively. These results suggest that GLUT3 is the major glucose transporter in the testes and that abdominal testes may increase the uptake of glucose into interstitial areas, leading to an increased risk of developing cancer.

Espermiogênese como ferramenta citotaxonômica para diferenciar Triatoma guazu e T. williami, espécies vetoras da doença de Chagas / Spermiogenesis as cytotaxonomic tool to differentiate Triatoma guazu and T. williami, vector species of Chagas disease

Os triatomíneos pertencem à ordem Hemiptera, subordem Heteroptera, família Reduviidae e subfamília Triatominae. Todas as 148 espécies são hematófagas estritas e potenciais vetoras do protozoário Trypanosoma cruzi, agente etiológico da doença de Chagas. Algumas espécies são extremamente semelhantes do ponto de vista morfológico, o que pode dificultar o trabalho dos programas de controle de vetores. Triatoma guazu e T. williami são consideradas como espécies irmãs e não podem ser diferenciadas por diferentes abordagens, como análises morfométricas, isoenzimáticas e cromossômicas. Assim, o presente trabalho analisou as células haplóides durante a espermiogênese, com o objetivo de auxiliar na diferenciação desses vetores. A análise das espermátides de T. guazu e T. williami permitiu diferenciá-los, pois T. williami apresentou apenas um filamento heteropicnótico nas espermátides iniciais que foi parcialmente mantido durante o alongamento celular e T. guazu apresentou dois filamentos heteropicnóticos periféricos que se uniram durante o alongamento celular, dando origem a uma espermátide alongada totalmente heteropicnótica. Assim, ressaltando a importância dessa ferramenta na diferenciação de espécies relacionadas. No entanto, sugerimos que cruzamentos híbridos experimentais devem ser realizados entre esses triatomíneos, com o intuito de confirmar o status específico desses insetos vetores da doença de Chagas.(AU)

Triatomines belong to the Hemiptera order, Heteroptera suborder, Reduviidae family and Triatominae subfamily. All the 148 species are haematophagous strict and potential vectors of the protozoan Trypanosoma cruzi, etiologic agent of Chagas disease. Some species are very similar from a point of view morphological, what can difficult the work of vector control programs. Triatoma guazu and T. williami are considered sister species and cannot be differentiated by many approaches such as morphological, isoenzyme and chromosome analysis. Thus, the present study examined the haploid cells during spermiogenesis, aiming to assist the differentiation of these vectors. The analysis of spermatids of T. guazu and T. williami allowed to differentiate them because T. williami presented only one heteropyknotic filament in the early spermatids which was partially maintained during cell elongation and T. guazu presented two peripheral heteropycnotic filaments that have joined during cell elongation, leading to a elongated spermatid fully heteropycnotic. Thus highlighting the importance of this tool in the differentiation of related species. However, we suggest that experimental hybrid crosses should be made between these triatomines, in order to confirm the specific status of these insect vectors of Chagas disease.(AU)

Histological Alterations in the Rabbit Epididymis Induced by Sildenafil Overdosing.

Background: Sildenafil is used for the treatment of erectile dysfunction and is helping millions of men around the world to achieve and maintain a long lasting erection. The histological alterations in the genital system epithelial lining due to sildenafil overdoses intoxication has not yet been well documented. Aim: The present study was carried out to investigate the histological alterations induced by sildenafil overdoses in the epididymal epithelial lining. Methodology: Fifty adult male rabbits (Oryctolagus cuniculus) were subjected to sildenafil (0, 1, 3, 6, 9 mg/kg/day) for 5 days per week for 7 weeks. Samples from left and right proximal epididymis were applied to conventional histological techniques and subjected to histological examination. Results: Exposure to overdoses of sildenafil had provoked histological alterations in the epididymal tissue in the form of epididymal hyperplasia and dilated lumina. Mature spermatozoa were less frequent in the lumen of the epididymis than the control ones while spermatogenic cells, cellular debris and spermatid giant cell were seen in the lumen of the epididymis. Conclusion: The results of the present study confirms that sildenafil provoked alterations in the epididymal epithelial lining together with partial arresting spermatogenesis and impairing the spermatic cells differentiation towards maturation. The findings together might indicate an affect on male fertility induced by sildenafil overdoses.

Effect of caprine testicular extract on acrosome formation of spermatids of lead-injured mice / 解放军医学杂志

Objective To investigate the effect of caprine testicular extract on the acrosomal formation of spermatids of lead-treated mice, and find a theoretical and experimental basis for further research on the effects of caprine testicular extract on the reproductive system. Methods The study was carried out on thirty male adult ICR mice, which were randomly assigned into control group, model group and treatment group (10 each). The testis-injury model was reproduced by intragastric administration of lead acetate (30mg/kg, 0.1ml/10g). Animals in treatment group received intragastric administration of lead acetate and intraabdominal injection of 0.5ml caprine testicular extract at the same time for 21days, and the mice in control group received the same amount of distilled water by intragastric administration and 0.5ml distilled water by intraabdominal injection. By the end of the experimental period, the mice were sacrificed. The testes were fixed by cardiac perfusion and processed for light microscopic examination. The acrosomal formation in round spermatids was observed by PAS staining. Results Compared with the control group, the acrosomal formation in model group was blocked. Acrosomal vesicle was incomplete, the membrane was shrunken, rough and fuzzy. The acrosomal formation in treatment group was similar to that of the control group. The structure of the acrosomal vesicle was complete and the membrane was smooth. Conclusion Administration of caprine testicular extract may protect the acrosomal formation of spermatids from lead poisoning, and the normal structure and function of the acrosome are preserved. Thus, indepth study of the effects of caprine testicular extract may contribute to the treatment of male infertility caused by heavy metal-induced testicular damage, and offer an effective protective measure for the lead-exposed males.

The long-term excessive alcohol consumption on sperm quality and the impact of germ cell apoptosis / 中国医师杂志

Objective To investigate the long-term excessive drinking semen nitric oxide (NO) content in their sperm quality and spermatogenic cell apoptosis and infertility. Methods Nitrate reductase method was used to specific reduce the NO metabolites nitrate (NO3-) to nitrite (NO2-), which was used on behalf of the total NO level. Terminal deoxynucleotidyl transferase (TdT) and mediated nick end labeling (TUNEL) method and binocular optical microscope were used to detect and observe the rate of apoptosis of spermatogenic cells and the morphological structure. The SQA-V sperm quality automatic analyzer was used to measure sperm quality. Results In not drinking semen fertility group, NO content was (54.81±11.45)μmol / L, the rate of spermatogenic cell apoptosis was (4.52±1.23)%, sperm motility was (80.24±0.17)%, energy a+b (78.32±0.12)%, deformity rate (5.30±0.13)%, and long-term excessive alcohol infertility C group was [(128.83±22.73)μmol/L,(17.34±2.53)%,(51.18±0.58)%,(21.45±0.26)%,(21.12±3.24)%] respectively. Compared to a very significant difference (t＝10.04,17.38,6.69,15.59,17.02,P＜0.01) . In long-term excessive drinking group, the levels of NO and spermatogenesis cell apoptosis rate was significantly positive correlated (r＝0.93,P＜0.01).Apoptosis of spermatogenic cell nucleus chromatin was condensed in the formation of crescent-shaped perinuclear, nuclear was cleavaged to form apoptotic bodies. Conclusions In the long-term excessive drinking semen, NO content and spermatogenic cell apoptosis rate was increased, the sperm had poor quality. The results showed that the long-term excessive drinking can cause germ cell apoptosis in male infertility and promote the body to overproduce NO, whichmay be one of the reasons for male infertility.

Influence of aoustic contrast agent with diagnostic ultrasound on rabbits' spermatogenic cell / 中华超声影像学杂志

Objective To study the impact of ultrasound contrast agent with different doses or imaging modes on rabbit spermatogenic cell in the diagnosis of radiation dose.Methods Thirty-three New Zealand white rabbits were randomly divided into 3 groups.In group Ⅰ ,three minutes gray scale ultrasound radiation was performed for three rabbits.In group Ⅱ, SonoVue was bolus injected from ear vein for eighteen rabbits.The rabbits in this group were divided into 6 sub-group according to different doses and different sampling times, the single injections of SonoVue with dosage of 0.1 ml/kg(A1, A2) or 1 ml/kg (C1 ,C2) and repeated injections with dosage of 0.1 ml/kg interval 15 min (B1 ,B2) were taken.In group Ⅲ ,twelve rabbits were bolus injected with SonoVue and then the agent bubbles were blasted when the SonoVue suffused the whole testis.The rabbits in this group were divided into 4 sub-groups according to different doses and different sampling times, and the dosages of SonoVue were 0.1 ml/kg(D1, D2) and 1 ml/kg(E1,E2).Testis were drew immediately or 24 hours later for the observation under light microscope and transmission electron microscope.Results Apoptotic cells in group Ⅲ were more than those in group Ⅰ and Ⅱ ( P ＜0.05) ,while no significant difference of the total number of apoptotic cells between group Ⅰ and Ⅱ ( P ＞0.05).The appearances of focal ultra-structural damage were observed by transmission electron microscope in group C1, such as intercellular space widening and mitochondria swelling, and no distinct damage were observed in the other groups.The appearances of intercellular space widening, mitochondria swelling and cellular edema were observed by transmission electron microscope in group Ⅲ, and spotty necrosisetc was also observed in group D1 and E1.Conclusions Large dose of contrast agent could cause germ cells slight instantaneous effects, while conventional dose had no effect.Contrast agent blasting may cause germ cells unrecoverable damage.

Effect of Testosterone Propionate on Apoptosis of Rat Germ Cells / 天津医药

Objective: To investigate the effects of the exogenous testosterone propionate on apoptosis of rat germ cells and the mechanisms thereof. Methods: Thirty 35-day-old male SD rats were randomly divided into experimental group and the control group. The rats in experimental group were injected (i.m.) testosterone propionate and the control group with an equal volume of saline. By using terminal deoxynueleotidy transferase nediated dUTP nick-end labeling (TUNEL), flow cytometry (FCM), radioimmunoassay (RIA) and electron microscopy, the quantity and quality of apoptosis of germ cells were evaluated. Results:(1) Compared with the control, the apoptotic number of rat germ cells was increased in the experimental group, especially the primary spermatocyte. The apoptotie rate was 11.3% detected by FCM in experimental group,while 3.6% in the control group (P ＜ 0.01). (2) The percentages of 1C were 21.8% in experimental group and 33.8% in control group (P ＜ 0.01).The percentages of 2C were 52.6% in experimental group and 37.1% in control group (P ＜ 0.01). (3) The serum levels of testosterone were (3 486.8±333.3) ng/L in experimental group and (846.9±167.5) ng/L in control group (P ＜ 0.01). The serum levels of follicle-stimulating hormone (FSH) were (2.5±0.8) IU/L in experimental group and (5.2±1.7) IU/L in control group (P ＜0.01). Conclusion: The exogenous testosterone propionate might induce apoptosis of germ cells by retroinhibition of the hypothalamie-pituitary-gonadal axis, thus having contraceptive effects.

The extract of "shoe flower" (Hibiscus rosea sinensis L) leaves inhibit the spermatogenesis of ddy strain mice.

This study was conducted in order to develop male contraception from plants, namely the "shoe flower" (Hibiscus rosea sinensis L) leaves. The objective of this study was to find out whether the extract of "shoe flower" leaves could inhibit the process of spermatogenesis on ddy strain mice. This research was performed in 3 groups and each group consisted of 8 mice. The control group was given 1% carboxy methyl cellulose (CMC) in 0.5 ml aquabides. The treatment group I was given the extract of "shoe flower" leaves 700 mg/kg BW and 1% CMC in 0.5 ml aquabides, and the second treatment group was given the extract of "shoe flower" leaves, 800 mg/kgBW and 1% CMC in 0.5 ml aquabides. The treatment were given for 40 days in accordance with the spermatogenesis cycle. Then, the production of histological slides of the mice testis and the observation of the slides using light microscope with magnification of 100x and 400x were done. Further, counting of the spermatogenic cells was done. At last the pictures of seminiferous tubulus cross-section of the three groups which consisted of spermatogenic cells were taken through light microscope with magnification of 100x and 400x using Fuji camera and Fuji film, 200 ASA. The results showed significant differences between the control, treatment I, and treament II group. There were decreased numbers of spermatogonia, pachyten primary spermatocytes and spermatids in treatment groups (P<0.01). The result of this study showed that the extract of "shoe flower" (Hibiscus rosea sinensis L) leaves, inhibited the process of spermatogenesis of ddy strain mice. It is hoped that the result of this study can be developed into a male contraception.

Ultrastructural Aspects of Spermatids in Isogenic Black Mouse C57BL6J / Aspectos Ultraestructurales de las Espermátidas en el Ratón Negro Isogénico C57BL6

The objective of this study was to describe some changes in spermatids differentiating events during spermiogenesis, leading to spermatozoa final formation. The animals were sacrificed with ethylic acid inhalation and, then, testis fragments were collected, fixed in Karnovsky and post-fixed in osmium tetroxide. After dehydration, the sections were stained with uranyl acetate 2%. It was observed in early spermatids at Golgi phase, formation of acrosomal granule contained in the acrosomal vesicle, spheroidal nucleus with rugous reticulum around it, and Golgi apparatus made up of lamellae and vesicular structures. At cephalic hood phase, the granule suffers flattening and the marginal fossa is formed. In both phases, it was possible to observe the presence of multivesicular body. At acrosomal phase, the acrosomal system is set up, and several enzymes of endoplasmatic reticulum as well chromatoid body are observed in the cytoplasm. At the last phase, that is, maturation, the nucleus becomes more elongated, the endoplasmatic reticulum regresses, the Golgi apparatus approaches to the flagellum and mitochondrias align next to it.

El objetivo del estudio, fue describir algunas alteraciones producidas en la espermiogénesis, en los eventos que diferencian a las espermátidas y que llevan a la formación final de los espermatozoides. Los ratones fueron sacrificados con inhalación de éter etílico y, enseguida, se recolectaron fragmentos de testículos, los que fueron fijados en Karnovsky y tetróxido de osmio. Después de la deshidratación, los cortes fueron teñidos con acetato de uranilo al 2 %. En espermátidas jóvenes en la fase de Golgi se observan: formación del gránulo acrosómico contenido en la vesícula acrosómica, núcleo esferoidal con retículo rugoso a su alrededor, y aparato de Golgi formado por lamelas y estructuras vesiculares. En la fase de capuz cefálico, el gránulo sufre achatamiento y se forma la fosa marginal. En estas dos fases fue posible observar la presencia del cuerpo multivesicular. En la fase acrosómica se establece el sistema acrosómico, distingiéndose en el citoplasma muchas vesículas de retículo endoplasmático y cuerpo cromatoide. En la última fase, la de maduración, el núcleo se torna más alargado, el retículo endoplasmático retrae, el aparato de Golgi se aproxima al flagelo y las mitocondrias se alinean a lo largo de él.

PC3 prostasome promotive effects on fresh or cryopreserved human spermatozoa in swim-up medium / 中华泌尿外科杂志

Objective To investigate the motility stimulant effects of prostasome like granules from the PC3 prostate cancer cell line (here called PC3 prostasome). Methods Fresh or cryopreserved human spermatozoa in Earle's balanced salt solution (EBSS) supplemented with human serum albumin (HSA,10 mg/ml) or PC3 prostasome (0.10 mg/ml or 0.25 mg/ml)after swim up to compare the difference of motility or the recovery of motility. Results For the fresh samples within different time of incubation or different parameters in one hour incubation,addition of PC3 prostasomes 0.10 mg/ml was superior to HSA in sperm motility stimulation ( P

Abnormalities in the fine structure of the spermatids of rats injected with cadmium.

The degenerative changes in the spermatids as measured by changes in fine structure abnormalities increased with time following injection of Cd2+ into rat testis. The spermatids in the twelve hours group appear as peculiarly club shaped and elongated structures with one or two small but perceptible vacuoles. The subacrosomal area and the space between the nucleus and the middle piece are seen abnormally dilated. In the 30 day group, the central filaments are the most susceptible unit of 9+2 axoneme complex. The plasma membrane, the cytoplasmic matrix, the mitochondria of the middle piece and the fibrous sheath appear shrunken, discontinuous and degenerative.