Isolation of germ cells from human testicular tissue for low temperature storage and autotransplantation

OBJECTIVE: To develop a new protocol for conserving fertile potential in men undergoing sterilizing chemotherapy by low temperature banking of germ cells which can be returned to the patient's testes after thawing. DESIGN: Isolation of human and murine germ cells for comparing cellular viability after cooling to liquid nitrogen temperatures by the use of different cryoprotective agents and for infusion into the testis. SETTING: Laboratory research environment. PATIENT(S): Men undergoing routine surgery in a urology department. INTERVENTION(S): Testicular biopsy. MAIN OUTCOME MEASURE(S): Cellular viability and infusion of seminiferous tubules. RESULT(S): After isolation using a two-step enzymatic disaggregation protocol, 66% to 87% of germ cells from human and murine specimens, respectively, were still viable. Cell survival was similar in four commonly used cryoprotective agents after cooling to liquid nitrogen temperatures. Seminiferous tubules infused by back flow with dye solution via the rete testis were filled with an efficiency of 55%. CONCLUSION(S): Judging from the high viability of unfractionated germ cells, it is feasible to isolate germ cells from testicular biopsies for low temperature banking with the aim of attempting to restore fertility after iatrogenic sterilization.     

Testicular involution in elderly men: comparison of histologic quantitative studies with hormone patterns

An endocrinologic and quantitative histologic study was carried out in 64 elderly men who underwent orchidectomy owing to prostatic carcinoma. The men were classified into age groups (decade of life), and each group was subdivided into group A (testes with complete spermatogenesis in most tubules) and group B (testes showing maturation arrest of spermatogenesis in most tubules). Up to 80 years of age, men of group A showed hormone levels and testicular parameters similar to those of young control men. From 50 to 60 years of age, men of group B showed a significant decrease in testicular volume, tubular volume, tubular length, number of germ cells, Sertoli cells and Leydig cells per testis, and plasma testosterone levels, whereas the tunica propria thickness and plasma levels of both follicle-stimulating hormone and luteinizing hormone were increased.     

Identification of seminiferous tubule aberrations and a low incidence of testicular microliths associated with the development of azoospermia.

OBJECTIVE: To evaluate the use of percutaneous testicular sperm aspiration in the assessment of azoospermia and its association with seminiferous tubule microliths. DESIGN: Case report. SETTING: Tertiary care fertility center in a university hospital. PATIENT(S): Male undergoing infertility evaluation. INTERVENTION(S): Testicular biopsy and percutaneous testicular aspiration. MAIN OUTCOME MEASURE(S): Serum hormone analysis, sperm concentration in semen, spermatogenesis in samples from testicular biopsies and aspirations, and microlith composition. RESULT(S): A patient presented for infertility evaluation with a history of severe oligospermia that progressed to azoospermia. The serum testosterone concentration (357 ng/dL) and LH concentration (9.2 mIU/mL) were normal and the serum FSH concentration (18.3 mIU/mL) was elevated. Testicular biopsy results indicated spermatogenic hypoplasia with limited spermatozoa. Seminiferous tubules obtained by percutaneous testicular aspiration were structurally aberrant, with multiple diverticula. Microliths averaging 120 microm in diameter were observed within and blocking the seminiferous tubules. The microliths were composed of calcium phosphate (hydroxyapatite) in both the core and peripheral regions. Electron microscopy revealed a high degree of collagen-like material within the peripheral zone. CONCLUSION(S): The presence of seminiferous tubule microliths is associated with the development of azoospermia. In patients with a low incidence of seminiferous tubule microliths and aberrant seminiferous tubule architecture, percutaneous testicular aspiration may provide a diagnostic advantage over testicular biopsy.     

Male hyperprolactinemia:effects on fertility.

Male hyperprolactinemia was detected in 4% (7 of 171) of infertile men. In seven patients with excessive serum prolactin concentrations, the clinical manifestations were infertility, hypogonadism, impotence, and galactorrhea and the etiologic factors were pituitary adenoma, hypothalamic dysfunction, drug use, and idiopathic. The testes and prostate were small or normal and the semen analysis revealed low semen volume, normal or low sperm count, and normal or impaired sperm motility. The testicular biopsy showed normally preserved seminiferous tubules with normal or decreased spermatogenesis and damaged or fibrotic seminiferous tubules among normal ones. Patients with hyperprolactinemia were investigated by sellar polytomography, visual field examinations, and hormone assays. Treatment with bromocriptine (Parlodel) gave satisfactory results in all patients. The use of bromocriptine with human menopausal gonadotropin and human chorionic gonadotropin was beneficial in treating hypogonadotropic hypogonadism with hyperprolactinemia.     

A percutaneous large-needle aspiration biopsy technique for histologic examination of the testis in infertile patients

OBJECTIVE: To describe a relatively new percutaneous large-needle aspiration biopsy technique for histologic examination of the testis in infertile patients. DESIGN: Retrospective analysis of clinical and pathologic data. SETTING: Clinical and academic research environment. PATIENT(S): Sixty-six infertile patients who underwent testicular biopsy. INTERVENTION(S): Local anesthesia was induced through spermatic cord block with lidocaine, and a relatively large needle (usually 18- or 20-gauge) was introduced percutaneously into the testicle without a scrotal incision. MAIN OUTCOME MEASURE(S): The number of seminiferous tubules per histologic section of each testicular biopsy sample. RESULT(S): A mean of 74 seminiferous tubules were obtained in the histologic sections of each biopsy sample. This number varied according to the size of the needle used; it was 24.7 when a 21-gauge needle was used, 56.2 when a 20-gauge needle was used, and 103 when an 18-gauge needle was used. The biopsies were performed in the office. No significant hematomas occurred, no antibiotic prophylaxis was prescribed, and no postbiopsy medical or pharmacologic interventions were required. CONCLUSION(S): Tissue specimens as large as those obtained with open surgical biopsy can be obtained from the testicles of infertile patients with the use of a percutaneous technique that is easier, less costly, and safer than any previously reported.     

促黄体素释放激素对大白鼠睾丸的组织学和组织化学观察

用81只雄性大白鼠分为9组,分别给予LHRH-A_3 0.02μg、2μg 及200μg 三种剂量注射2天,20天及60天。在组织学上见200μg 注射20天后,睾丸开始出现抑制退化现象。200μg 注射60天后,则抑制现象更加明显。睾丸重量下降,曲细精管萎缩,生精细胞疏松脱落,精子细胞溶解,精母细胞膨大变形,形成严重生精障碍。在组织化学上,注射LHRH-A_3 200μg 60天后,可见睾丸的新陈代谢受到影响,物质的吸收及转运出现抑制,生精上皮有一定损伤,睾酮的合成增强但分泌受到障碍。提示注射LHRH-A_3 200μg 60天后,对大鼠精子生成和睾酮的分泌发生明显的抑制作用。     

Complete Androgen Insensitivity Syndrome with a Sertoli-Leydig Cell Tumor

BackgroundThe complete androgen insensitivity (testicular feminization) syndrome was described in phenotypic females with 46XY karyotype, presenting with primary amenorrhea, adequate breast development, and absent or sparse pubic or axillary hair. Gonads consist usually of seminiferous tubules without spermatogenesis.CaseWe report the case of a 15-year-old girl with testicular feminization since age 4. She was admitted to surgically remove the testes from the inguinal canal. Microscopic examination of the left testis revealed a Sertoli-Leydig cell tumor and the draft of fallopian tube with adjacent seminiferous tubules.Summary and ConclusionThese patients have a 5%-10% risk of developing germ cell tumors. Sertoli-Leydig cell tumors are uncommon neoplasms, usually benign in testicular feminization syndrome.     

Antigens of the basement membranes of the seminiferous tubules induce autoimmunity in Wistar rats

A preparation enriched in basement membranes from seminiferous tubules was isolated from rat testes (STBM) and injected with complete Freund's adjuvant into Wistar rats. In 60% of the animals a mild multifocal orchitis was observed. In damaged areas, perivascular and peritubular mononuclear cell infiltrates and different degrees of cell sloughing of some seminiferous tubules were observed. Electron microscopy revealed focal thickenings and delamination of the basement membrane of the seminiferous tubules as well as vacuolization of Sertoli cell cytoplasm. Using immunofluorescence discontinuous linear deposits of IgG were detected along the seminiferous tubular wall. Moreover, the same pattern of immunofluorescence was observed when the IgG eluted from the testes of the immunized rats was layered on sections of normal rat testis. Circulating antibodies to STBM were detected using passive haemagglutination in approximately 45% of the immunized rats, with titers ranging from 1:20 to 1:80. Leukocyte migration was inhibited when the spleen cells of the immunized rats were incubated with antigens from the basement membrane of seminiferous tubules, whilst a negative reaction was obtained when the soluble fraction of testis homogenate was used.     

Testicular sperm extraction using microdissection for non-obstructive azoospermia

Background : Men presenting with non-obstructive azoospermia (NOA) caused by germinal failure can now be treated in some cases using testicular sperm extraction (TESE) and intracytoplasmic sperm injection (ICSI). However, TESE is a blind procedure that does not identify the focal sperm-producing areas until excision of the testicular tissue. Microdissection TESE, which is the only method available for obtaining excised dilated seminiferous tubules under the operating microscope, improves sperm yield with minimal tissue excision in NOA patients.
Methods and Results : We performed this procedure on 16 NOA patients. All subjects underwent a microdissection TESE on the right testis, and triple biopsy on the left testis in consecutive fashion in order to compare the efficacy of microdissection TESE with that of a standard biopsy. Although dilated seminiferous tubules were presented in all patients, spermatozoa were retrieved in only a single patient by microdissection TESE. Furthermore, spermatozoa could not be identified by standard biopsies.
Conclusion : In this series, microdissection TESE did not contribute to spermatozoa recovery in NOA patients. Further study is needed in order to arrive at a reliable assessment of microdissection TESE relative to a standard multiple biopsy in cases of NOA. (Reprod Med Biol 2002; 1 : 31–34)     

Chemical sterilization: effects of a single high dose of 3-chloro-1,2-propanediol on the testes and epididymides of dog.

A single high dose (70 mg/kg) of 3-chloro-1,2-propanediol (alpha-chlorohydrin) caused pathological degeneration in the testes of dog when examined after 33 days. The seminiferous tubules were depleted of spermatogenic elements. Epididymal epithelium was regressed, the lumen was empty and the passage through the duct was unimpaired. Alpha-chlorohydrin inhibited the synthesis of RNA and sialic acid of the testes and epididymides. A single high dose of alpha-chlorohydrin increased the total cholesterol/g of wet testis tissue which could be correlated with the testicular atrophy. Antiandrogenic nature of the compound has been discussed.     

Testicular sperm extraction by Trucut needle and milking of seminiferous tubules: a technique with high yield and patient acceptability

OBJECTIVE: To assess the sperm yield and patient acceptability of Trucut needle testicular biopsy followed by seminiferous tubule milking. DESIGN: Prospective case analysis. SETTING: The Regional Fertility Center, Royal Maternity Hospital, Belfast, Northern Ireland, United Kingdom. PATIENT(S): Forty-one males with obstructive azoospermia (normal testicular volume and FSH and LH levels). INTERVENTION(S): Trucut needle testicular biopsies under local anesthetic with milking of the seminiferous tubules. MAIN OUTCOME MEASURE(S): Quantitation of sperm retrieved per biopsy core and patient follow-up by questionnaire. RESULT(S): A mean of 105,634 sperm (range, 5,000-427,800) were retrieved, and the mean biopsy weight was 9.17 mg. Twenty-six subjects found the biopsy painless and 15 were pain-free after biopsy. CONCLUSION(S): The Trucut needle can be used in combination with seminiferous tubule milking to obtain large numbers of sperm in men with obstructive azoospermia.     

Effects of cadmium chloride on the testis and sex accessory glands of the Indian palm squirrel, Funambulus pennanti (Wroughthon).

A single subcutaneous injection of cadmium chloride caused reversible destruction in the testis of palm, squirrel, Funambulus pennanti. Marked regression in testicular weight and seminiferous tubule diameter associated with complete necrosis of germinal epithelium was observed after 1, 2, 3, 5 and 7 days of cadmium administration. A significant increase in the concentration of testicular cholesterol was observed after 5 days treatment. Decrease in the citric acid concentration of prostate gland was associated with the degenerative changes of the spermatogenic elements. Reduced fructose concentration in the seminal vesicle and prostate of cadmium treated animals indicate an inhibition of androgen production. Decreased androgen production was also reflected by the atrophic seminal vesicles, prostate and epididymides. These effects were reversible and about 70% seminiferous tubules showed normal spermatogenesis. The cholesterol, fructose and citric acid concentration of testis and sex accessory glands returned to subnormal levels after 45 days of cadmium chloride treatment. It is suggested that the degenerative effects of cadmium chloride are dose, time and species dependent.     

Temporal adaptation in the testes of rat administered single dose Momordica charantia for three interrupted spermatogenic cycles: Cytometric quantification

Introduction

Although unrelenting attempts have been made during the past five decades in understanding the three-dimensional morphology of the rats’ testis and the organizational arrangements of the seminiferous tubules, the findings have remained ambiguous relating the structure to its function.

Objective

This article investigates the effects of Momordica charantia (MC) on the testes of Sprague–Dawley (S–D) rats using stereological tools.

Methodology

A total of 50 male S–D rats, weighing 110–214 g were allocated randomly into three main groups I to III. Groups I and II were further divided into 10 subgroups of five rats. The duration of 6, 8, 16, 24, and 32 weeks of extract treatment represented 5 subgroups in Group I. While 8, 16, 24, and 32 weeks were 4 subgroups in Group II. Groups I and II received daily oral doses of 50 mg/100 g body weight of MC seed extract for the respective duration. Group III, (control) rats were fed an equal volume of physiological saline. The animals were sacrificed at the end of the experimental period and the testes were processed for cytometry. Stereological parameters estimated include: Testicular volume and weight, tubular diameter, cross sectional area of seminiferous tubules, number of profiles per unit area, length density and numerical density of the seminiferous tubules.

Result

Histomorphometric quantification showed a duration dependent decrease in the mean testicular volume and weight, seminiferous tubular diameter and cross sectional area of tubules; but an increase in the number of profiles per unit area, length density and numerical density of the seminiferous tubules. Those animals investigated for reversibility showed an appreciable recovery at discontinuation of the extract as testicular values compared similarly to their control counterpart.

Conclusion

The Cytometric data support a time-related deleterious effect on the testes of S–D rats treated with Momordica charantia seed extract.     

大鼠睾丸受氯化镉损伤后生精上皮恢复的观察

目的:探讨氯化镉(CdCl2)染毒大鼠致睾丸损伤后生精上皮能否恢复。方法:20只雄性SD大鼠随机分为对照组(A组)与3个CdCl2实验组(B、C、D组),每组5只动物。B-D组的动物分别腹腔注射CdCl21mg/kg,qd×14d。对照组腹腔注射等体积0.9%NaCl。染毒结束后,处死A和B组大鼠,C组经口服灌胃给予ZnCl2100mg/kg,qd×7d,D组不给予ZnCl2。C和D组染毒结束后再继续饲养2个月(恢复期),观察睾丸生精上皮的组织学改变,比较4组的异常曲细精管率。结果:B组部分曲细精管显示病理改变,B组的异常曲细精管率与A组的比较有显著升高(P<0.01)。恢复期后C和D组的曲细精管有明显恢复,大多数曲细精管显示正常曲细精管组织学图像,C和D组的异常曲细精管率与B组的比较均有显著降低(P<0.01)。C和D组之间的异常曲细精管率比较没有显著差异(P>0.05)。结论:在本实验条件下,CdCl2损伤后的大鼠睾丸,在自然状态下和给予ZnCl2,生精上皮均可得到显著恢复。     

自发雄性不育近交系MIJ大鼠形态学特征观察

目的:了解本实验室培育的自发雄性不育近交系MIJ大鼠的形态学特征。方法:分别取1月龄、2月龄、3月龄不育大鼠(不育组,A组)、近交系内正常大鼠(系内正常组,B组)、正常雄性Wistar大鼠(正常对照组,C组),观察生长发育情况,常规方法制备病理切片,观察睾丸、附睾等器官组织学变化;取骨髓,常规方法染色体制片,G显带,进行染色体分析。结果:不育组大鼠体重增长速度稍慢,生殖器官发育迟缓;1月龄时腹腔内隐睾;睾丸下降时间、精索长度、睾丸引带长度、睾丸系数、附睾系数均明显落后于其他二组;且睾丸生精上皮发育不全,附睾内精子数量明显减少;但染色体形态及数目均正常,未观察到畸变。结论:自发雄性不育近交系MIJ大鼠呈睾丸下降延迟、睾丸发育不全、少精等特征,可以作为人类男性不育研究的良好动物模型。     

Immunocompetent cells in human testis in health and disease

The authors have investigated lymphocyte subpopulations and macrophages in normal human testes and the testes of patients under investigation and treatment for subfertility. Specific monoclonal antibodies were used in an indirect immunoperoxidase technique. In normal tissues, T lymphocytes (Leu 4-positive cells) were present in the rete testis with a preponderance of cells of the suppressor/cytotoxic phenotype. In contrast, no lymphocytes were detected within the peripheral portions of the testis. Cells reacting with the anti-Leu M3 monoclonal antibody, which defines monocytes/macrophages, were detected in appreciable numbers in peripheral testis with a specific location around the seminiferous tubules. HLA-DR-positive cells (human leukocyte antigens--class II [DR] determinants of the major histocompatibility complex) also were identified and showed a similar pattern of distribution to that of the Leu-M3 positive cells. While no lymphocytes were seen in the normal peripheral testis, T lymphocytes were detected in testicular biopsies from subfertile patients. Suppressor/cytotoxic T cells (Leu 2a-positive) predominated in patients with oligozoospermia and obstructive azoospermia while T cells of the helper/inducer phenotype predominated in patients with unilateral testicular obstruction and in postvasectomy patients. Sperm antibody measurements correlated with these findings.     

Effect of protopanaxatriol saponin on spermatogenic stem cell survival in busulfan-treated male mice

Background and aims:  Panax ginseng C.A. Meyer is a medicinal herb widely used in Asian countries. Many of its pharmacological actions are attributed to ginsenosides (saponin). However, the pharmacological effects or functions of ginsenosides on mammalian spermatogenesis are unclear.
Methods:  In the present study study, we investigated the therapeutic and prophylactic effects of protopanaxatriol saponin (PT) on testicular organ weight and morphology, testicular germ cells, proliferation, differentiation and spermatogenesis after induction of toxicity by a chemotherapeutic agent, busulfan, in male mice.
Results:  Intraperitoneally (IP) busulfan treatment markedly decreased the organ weight of testis, caput and cauda epididymis. After the treatment, the testes had collapsed seminiferous tubules with incomplete spermatogenesis. However, a single dose of busulfan treatment followed by PT injection showed milder damage on seminiferous tubules than busulfan alone.
Conclusion:  These results suggest that PT is effective in recovery of the male reproductive organ, and induced an increase in the number and viability of germ cells overcoming busulfan toxicity. PT might have applications in the recovery of male infertility arising from azoospermia and oligospermia.     

Decreased expression of the heat shock protein hsp70-2 is associated with the pathogenesis of male infertility

Objective: To examine the expression of the heat shock protein hsp70-2, and the possible relationship with the pathogenesis of male infertility.

Design: Prospective study.

Setting: Reproductive testing laboratory in a university hospital.

Patient(s): Men undergoing testicular biopsy during an investigation of subfertility.

Intervention(s): Testicular tissues were obtained from biopsies of men undergoing infertility evaluation and subdivided into three groups: normal testes, maturational arrest and Sertoli cell-only syndrome. Immunostaining and Western blotting techniques determined expression of the heat shock protein hsp70-2

Main Outcome Measure(s): Expression of the heat shock protein hsp70-2 in the testes.

Result(s): The experimental data demonstrated that the heat shock protein hsp70-2 was expressed in the normal and maturation arrest testicular specimens. The heat shock protein hsp70-2 was strongly present in the cytoplasm of spermatocytes and spermatides in the adluminal compartment of the seminiferous epithelium in normal testis. However, maturation arrest testis tissue demonstrated light staining in spermatocytes and spermatides, and Sertoli-only specimens demonstrated no staining for the heat shock protein hsp70-2. The Western blotting data showed a 70-kDa heat shock protein in the normal and maturation arrest testicular tissues, but not in the Sertoli-only tissues.

Conclusions: These results suggest that the heat shock protein hsp70-2 is expressed in spermatocytes and spermatides in normal and maturation arrest tissues. However, the expression of the heat shock protein hsp70-2 was low in maturation arrest, and no heat shock protein hsp70-2 was demonstrated in Sertoli-only specimens. Therefore the decreased expression of the heat shock protein hsp70-2 is associated with the pathogenesis of male infertility.     

Isolation and partial characterization of an interleukin-1-like factor from rat testis interstitial fluid

Testicular interstitial fluid (ISF) was collected by in vivo perfusion of rat testes and analyzed for the presence of interleukin-1 (IL-1) activity by utilizing a murine thymocyte proliferation assay. IS obtained from nine rats were all positive with dose-response curves of IL-1 activity similar to those produced by rat testicular aqueous extracts, rat macrophage IL-1 and human recombinant IL-1 alpha. Chromato-focusing of pooled ISF revealed a single peak of IL-1 activity with an estimated isoelectric point of 6.1-6.3. HPLC size exclusion chromatography demonstrated two active peaks with apparent molecular ratios Mr of 15,000-18,000 and 5000-7000, respectively. The molecular properties of the 15,000-18,000 Mr component are very similar to those of an IL-1-like factor previously isolated from seminiferous tubules. Our results indicate that the testicular IL-1-like factor is secreted by the seminiferous tubules into the interstitial tissue. Its function in the testicular interstitium is unknown but it might be relevant for the tendency to testicular relapse of childhood lymphocytic leukemia.     

Optimum testicular aspiration technique for deoxyribonucleic acid flow cytometric analysis of spermatogenesis in primates (Papio anubis)

Deoxyribonucleic acid flow cytometry on testicular tissue is an established method to evaluate spermatogenesis. Needle aspiration is less invasive and permits sequential sampling as compared with open testis biopsy. Using seven primates, we examined the needle aspiration technique. There was no significant difference among locations aspirated; the Westcott needle (Bard Urological, Covington, GA) provided the most rapid analysis, and both the Biopty gun (Bard Urological) and Westcott needle were easy to use. Baseline means +/- SD for ploidy distributions were 1N: 65.73% +/- 7.1; 2N: 18.95% +/- 5.5; 4N: 11.4% +/- 2, Experimental data generated from primate models using testis aspiration and flow cytometry may elucidate human infertile conditions.