Immunohistochemical Localization of Enkephalins in Adult Rat Testis: Evidence for a Gonadotrophin Control

The recent detection of immunoreactive POMC derived peptides in the male reproductive tract raised the possibility that these hormones might regulate reproductive function. In addition, a variety of studies suggested that POMC derived peptides have paracrine effects in the testis. In this present study, after HCG addition to the organ culture medium, Sertoli cells cytoplasma became immunoreactive with the enkephalins antisera.     

Inhibitory Effect of LHRH and Its Agonists on Testicular Gonadotrophin Receptors and Spermatogenesis in the Rat

Treatment of adult male rats with the potent LHRH agonists [D-Leu⁶, des-Gly-NH₂¹⁰]LHRH ethylamide or [D-Ala⁶. des-Gly-NH₂¹⁰]LHRH ethylamide led to a marked reduction of testicular LH and prolactin receptor levels accompanied by decreased plasma testosterone levels and testis, seminal vesicle and prostate weight. Maximal inhibitory effects were seen at a dose of 50 ng. After a single injection, maximal inhibitory effects were seen at 2–4 days with return toward normal levels at later time intervals. Similar results were obtained with LHRH itself although 5 μg was required for maximal effects. When the LHRH analog was administered twice a week at the 100 ng dose, degenerative signs appeared in the seminiferous tubules after one week while at four weeks, degenerative changes were so advanced that almost all germ cells had disappeared leaving only Sertoli cells.     

Studies on Factors Governing Testicular Decent in the Rat – Particularly the Role of Gubernaculum Testis

In the rat the testicles are abdominal at birth: testicular descent and scrotal development are completed at about 40 days of age. To elucidate mechanisms of testicular descent the following operations were performed in newborn rats: Proximal and distal unlateral gubernaculotomy, and unilateral and bilateral orchidectomy. The operated rats were studied at various time intervals up to 100 days after the operations. Testicular descent was prevented if the distal part of the gubernaculum was cut, presumably because of interference with the growth of the vaginal processes. The gubernaculum does not pull the testicle down into the scrotum, since testicular descent occurred also after proximal gubernaculotomy. The gubernaculum is, however, necessary to anchor the descended testicle to the scrotal floor. Descent of epididymis took place also in the absence of a testicle on the same side. We propose that distal gubernaculotomy can be used in rats to produce an experimental model for studies of cryptorchism.     

Effect of Streptozotocin-Induced Diabetes on Insulin Binding Parameters in Adult Rat Testis

Summary:  Insulin binding parameters have been measured in testicular membranes of streptozotocin diabetic male rats. Insulin binding decrease was ascribed to the well-known depressing effect of diabetes mellitus on circulating luteinizing hormone (LH). Because both LH and insulin receptors are modulated by pituitary LH and because of their reduction in testes of diabetic rats, we conclude that Leydig cell dysfunction is a secondary disorder associated with this complex metabolic condition.
Zusammenfassung:  An den testikulären Membranen von männlichen Ratten wurden Insulinbindungsparameter untersucht, die mittels Streptozotocin in eine diabetische Stoffwechsellage gebracht worden waren. Die Verminderung der Insulinbindungskapazität wurde dem negativen Effekt des zirkulierenden LH auf den Diabetes mellitus zugeschrieben. Sowohl die LH- als auch die Insulinrezeptoren werden durch hypophysäres LH moduliert. Aus der Verringerung dieser Rezeptoren im Hoden der diabetischen Ratte wurde geschlossen, daß die Leydigzell-Insuffizienz ein sekundäres Pänomen dieser komplexen Stoffwechselstörung ist.     

Distribution of Lectin Binding in Rat Testis and Epididymis

Summary: Seven lectins (PNA, RCA I, SBA, Con A, WGA, UEA I, DBA) conjugated with rhodamine were employed to analyse the staining pattern of glycoproteins with varying sugar residues in the testis and epididymis of adult Wistar rats. Some lectins (UEA I, SBA, DBA) gave rather specific staining of the mature acrosome, while others (PNA, RCA I) showed affinity for the early stages of acrosome formation or had a wide affinity for germinal and non-germinal cells and structures (Con A, WGA). In the epididymis the sperm mass had a homogenous staining reaction with some lectins (PNA, RCA I, Con A, WGA, DBA) which also showed a rather strong reaction on the epithelial surface. It was concluded that this reaction is at least partially due to the secretory products synthetized by principal, apical, narrow and light cells of the epididymal epithelium. Some differences in the staining pattern of these cells were recorded indicating specialization of the cells for the production of distinct glycoproteins. The staining pattern of the interstitial and intertubular compartment of the testis and epididymis was also recorded. Zusammenfassung: Die Verteilung der Lektin-Bindung im Hoden und Nebenhoden der Ratte Sieben Lektine (PNA, RCA I, SBA, Con A, WGA, UEA I, DBA) wurden verbunden mit Rhodamin verwendet, um das Darstellungsmuster von Glykoproteinen mit verschiedenen Zuckerresten im Hoden und Nebenhoden erwachsener Wistar-Ratten zu analysieren. Einige Lektine (UEA I, SBA, DBA) gaben eine recht spezifische Darstellung des reifen Akrosoms, wohingegen andere (PNA, RCA I) eine Affinität zu den frühen Stadien der Akrosomformation zeigten oder eine breite Affinität zu Germinal- und Nichtgerminalzellen und -strukturen aufwiesen (Con A, WGA). Im Nebenhoden zeigte die Spermatozoenmasse ein homogenes Darstellungsmuster mit einigen Lektinen (PNA, RCA I, Con A, WGA, DBA), welche auf der epithelialen Oberfläche auch eine recht starke Reaktion aufwiesen. Es wurde daraus geschlossen, daβ diese Reaktion zumindest teilweise auf die sekretorischen Produkte, die in den Haupt-, Apikal-, Schmal- und Hellzellen des Nebenhodens synthetisiert werden, zurückzuführen ist. Einige Unterschiede im Darstellungsmuster dieser Zellen wurden aufgezeichnet, die die Spezialisierung der Zellen für die Produktion bestimmter Glykoproteine anzeigen. Das Darstellungsmuster des interstitiellen und intertubulären Anteils des Hodens und Nebenhodens wurde ebenfalls aufgezeichnet.     

LH/hCG Responsive Adenylyl cyclase in Rat Testis and Testes from Testicular Feminized Male (Tfm) Rats and Mice: Desensitization by Homologous Hormone

Testes of testicular feminized (tfm) rats and mice, as well as of normal male rats contain an LH/hCG responsive adenylyl cyclase. Basal, as well as hCG stimulated activities were higher in tfm rats and mice than in normal rats. The presence of an LH/hCG responsive adenylyl cyclase in the testis of tfm rats and mice shows that the greatly elevated LH levels present in males having this syndrome, giving 80–90% reduction in LH//hCG receptors, do not cause an uncoupling of the remaining receptors from the adenylyl cyclase. It also shows that androgens are not essential for coupling of the LH/hCG receptors to the adenylyl cyclase.
Injection of 200 IU of hCG into adult normal rats and tfm rats caused, after 48 h, a complete loss of LH/hCG stimulated adenylyl cyclase, whereas the FSH responsive adenylyl cyclase in both animal preparations was maintained. Desensitization of the LH responsive adenylyl cyclase by hCG in normal rats, confirms previous studies showing lack of hCG stimulated cyclic-AMP secretion after a comparable dose of hCG in vivo. Similarly, hCG (50 IU) caused a transient loss of LH/hCG responsive adenylyl cyclase in tfm mice, with a complete disappearance of response after 24 h. At 48 and 72 h after injection of hCG the response gradually returned to normal. The fact that hCG caused a complete desensitization of the LH/hCG responsive adenylyl cyclase in both tfm rats and mice, proves that androgen receptor mediated events are not involved in hCG desensitization of the adenylyl cyclase in Leydig cells.     

Regional distribution of opioidergic nerves in human and canine prostates

The regional distribution of opioidergic nerves in the juvenile and adult human prostate and in the adult canine prostate has been studied immunohistochemically using well-characterized polyclonal antisera against multiple opioid peptides. Nerves displaying immunoreactivity (ir) for the proenkephalin (PRO-ENK) derivatives met-enkephalin (ME), leuenkephalin (LE), octapeptide, and heptapeptide (ordered in decreasing frequency) were present in the dorsolateral stroma of human prostate. In canine prostate, the situation was similar, but the number of opioid-ir nerve fibers was lower than in human prostate. In both species, staining for the prodynorphin (PRO-DYN) derivatives dynorphin A and alpha-neoendorphin or staining for the pro-opiomelanocortin (POMC)-derived beta-endorphin was not visualized. In addition to their presence in nerve fibers, PRO-ENK-ir opioids (octapeptide) occurred in intrinsic ganglionic cells situated in the capsule. Octapeptide but not LE-ir fibers supplied stromal blood vessels. The periurethral region and tissue adjacent to the ejaculatory ducts appeared to be devoid of opioid-ir innervation. We conclude that endogenous opioids apparently exclusively derived from PRO-ENK may fulfill important comessenger functions in the fine regulation of prostatic stromal tonus and of local vascular perfusion.     

Albumin Localization in the Testis of Adult Golden Hamsters by Use of Immunohistochemistry: Die Lokalisation von Albumin im Hoden adulter Goldhamster mit Hilfe der Immunhistochemie

There is an increasing interest in different biochemical roles of albumin (Alb) in respect to testicular function. For this reason, Alb was localized by the peroxidase-antiperoxidase technique in the testis of adult golden hamsters. A strong Alb immunoreactivity occurred in the interstitial space and on the surface of Leydig cells. A few Sertoli cells showed a strong Alb response under the appearance of so-called leakages traversing Sertoli-Sertoli cell junctions. Spermatogonia reacted strongly, while spermatids and sperms showed rather a mild, or moderate response. Spermatocytes remained unstained. When similar maturation stages of seminiferous tubules were compared, the number of reacting cells varied strikingly. Our results support the assumption that Alb or a protein with an Alb-like immunoreactivity exerts diverse functions of the testis.     

未成熟大鼠睾丸单侧扭转后对健侧血流和组织学的影响

目的:观察未成熟大鼠睾丸单侧扭转后对健侧睾丸血流供应和组织学的影响,并比较不同处理方法的疗效。方法:建立Wistar3周龄大鼠左侧睾丸扭转模型,分别建立对照组、扭转组、扭转复位组和扭转切除组,每组10只。彩色多普勒测量各组术前、术后8h(即扭转复位或切除术后2h)、12h、24h、72h右侧睾丸动脉收缩期最大血流速度,并于对照组和扭转组术后2h,扭转复位组和扭转切除组第1次术后12h各取2只大鼠右侧睾丸进行组织病理学观察。各组喂养至9周龄时分别取右侧睾丸进行组织学观察及检测各组大鼠右侧睾丸的生精小管直径(STD)、生精上皮细胞计数(CMSE)和睾丸活检评分(TBS)。结果:①未成熟睾丸左侧扭转后,右侧睾丸的血供呈持续性增加。②扭转组、扭转复位组和扭转切除组右侧睾丸均有不同程度的间质水肿和超微结构改变。③9周龄时扭转组、扭转切除组右侧睾丸重量均较对照组显著增加(P<0.01);各组大鼠STD、CMSE、TBS均无显著性差异(P>0.05)。结论:未成熟大鼠睾丸单侧扭转后可引起对侧睾丸的血供增加和组织学改变,轻微损伤后扭转复位和睾丸切除预后效果相似。     

Kinetic Properties of Hexokinase of Germ Cells in Rat Testis

The Michaelis-Menten constants (Km) of glucose, fructose, galactose, 2-deoxy-D-glucose and ATP as substrate for hexokinase of spermatocyte, spermatid and cauda epididymal spermatozoa extracts were measured. The Km value of glucose was very similar for all three germ cells. It was also true for all other substrates. The affinity of glucose for this enzyme was the highest while that of fructose was the lowest. The Km values were in general agreement with characteristics of hexokinase extracted from other tissues of rat. The Vmax values were also determined. The Vmax ratio of fructose to glucose showed the highest values of 1.1. The Vmax ratios of other substrates to glucose were below 1.0. The results suggest that hexokinase in the germ cells is similar to that in other tissues in its kinetic properties.     

Incorporation and Intracellular Hydrolysis of 131 I-Albumin Injected in Rat Testis

A method of cell separation by enzymatic attack and filtration was applied after injecting the testes. The results indicated that in the case of the interstitial injections, the fraction rich in interstitial cells captures from 30 to 50 times more RISA than that rich in germinal cells, and from 10 to 20 times more than that rich in Sertoli cells. Following the intratubular injection, radioactivity incorporation was greatly reduced and there were no differences in RISA (radioactivity/mg of protein) among the cell fractions. It was concluded that Sertoli cells do not have in its apical side a phagocytic activity comparable to that of the interstitial macrophages and possibly they incorporate only a small amount of tracer by a process of moderate pinocytosis.     

Effect of Fetal Irradiation on Testicular Receptors and Testosterone Response to Gonadotrophin Stimulation in Adult Rats

The amount of [125I]human chorionic gonadotrophin bound by testicular membrane preparations from adult male rats with germinal cell aplasia induced by x-irradiation in utero was significantly reduced in comparison to that of normal males. The in vivo serum testosterone response to an injection of human chorionic gonadotrophin was significantly decreased in the x-irradiated rats. Paradoxically, the in vitro maximal secretory response to gonadotrophin stimulation was markedly increased. It is concluded that a decrease in the number of receptors on Leydig cells subjected to chronically elevated serum levels of LH does not limit the capacity of the testis for steroid biosynthesis.     

Preliminary results on the clinical efficacy and safety of androgen inhibition by an LHRH agonist alone or combined with an antiandrogen in the treatment of prostatic carcinoma

We have used the paradoxical antigonadal effects of LHRH agonists as a chemical castration in advanced prostatic cancer. We report early results of a phase II study on the clinical efficacy of the LHRH agonist D-Ser (TBU)6, des-Gly-NH2(10) LHRH administered to patients with stage D prostatic carcinoma. Following dose-range finding studies using either intranasal (IN) (200 micrograms twice/day or 500 micrograms twice/day) or subcutaneous (SC) administration (50 micrograms once/day, we developed a sequential combination of SC (500 micrograms three times/day for seven days) and IN regimen that was administered for 3 to 16 months to a group of 23 patients with stage D prostatic carcinoma. Initiation of therapy was associated with a clinical flare in one patient during the first week of treatment. Mean serum testosterone levels were already decreasing at one week and remained inhibited to levels inferior to 1 ng/ml after the first four weeks of treatment. Overall assessment shows that within the first six months of treatment, 26% patients were improved, 39% were stabilized, and 35% were nonresponders. Fourteen patients were followed during the next six months: 29% continued to respond, 29% escaped, 21% remained stable, and 21% were nonresponders. Histologic studies from castrated patients showed changes in spermatogenesis correlating to the degree and duration of suppression of testicular steroidogenesis without signs of toxicity. Preliminary observations on the combination of the pure antiandrogen RU 23908 with Buserelin (n = 5) or castration (n = 3) suggest that the addition of an antiandrogen does not seem to improve the patients nonresponding to other hormonal suppressive therapy (Buserelin) administered before (n = 3) or concomitantly with the antiandrogen (n = 2). Three relapsing castrate patients responded to the antiandrogen, but the response was temporary in two (eight to nine months of therapy). No side effects other than hot flashes and decreased potency are related to LHRH agonist alone or to the low-dose antiandrogen. Multicenter trials will be necessary to delineate the place of LHRH agonist alone or LHRH agonist combined with an antiandrogen in the treatment of prostatic cancer.     

Immunohistochemical Detection of Prolactin and its Receptors in Human Testis/Immunhistochemische Bestimmung von Prolaktin und seinen Rezeptoren im menschlichen Hoden

Prolactin (PRL) and PRL receptors were localized by light microscopic immunohistochemistry in the human testes on interstitial Leydig-like cells. Similar stainings were obtained with both monoclonal antihuman PRL antibodies and polyclonal antibodies raised against rabbit mammary gland PRL receptors. Moreover, the detection of PRL binding sites by radio-receptor assay on the same samples gave a confirmation of these results. This suggests that PRL might act directly on Leydig cells in the man, in the same way as in the rodent.     

大鼠睾丸间质tau蛋白和微管结合蛋白表达的变化

目的 :检查老龄和青年大鼠睾丸间质组织中tau蛋白和微管结合蛋白的表达情况 ,籍此评价老龄大鼠睾丸衰退的发生机制。　方法 :应用二步法免疫组化技术检查 6月龄SD雄性大鼠 (10只 ,青年组 )和 2 8月龄同品系大鼠 (10只 ,老龄组 )睾丸组织抗Tau蛋白和抗微管结合蛋白的抗体免疫反应情况。　结果 :老龄大鼠睾丸间质组织中tau蛋白免疫反应阳性细胞数明显高于青年组 (P <0 .0 0 1) ,而青年组大鼠睾丸间质组织中微管结合蛋白免疫反应阳性细胞数明显高于老龄组 (P <0 .0 1)。　结论 :tau蛋白和微管结合蛋白免疫反应阳性细胞数量的变化可能与睾丸衰退有密切关系     

Effects of castration, sex steroids, LHRH and glucocorticoids on LHRH binding in the anterior pituitary of male rats

In the present study we have examined the effects of gonadotrophin releasing hormone (LHRH), sex steroids and glucocorticoids on the binding of LHRH to receptors in the pituitary of male intact and castrated rats. In intact rats, LHRH (10 μg/day) treatment for 11 days caused a significant increase in LHRH binding, whereas testosterone (500 μg/day) or oestradiol (50 μg/day) were inhibitory. 17-hydroxyprogesterone and dexamethasone were without effects. In castrated rats, LHRH caused a marginal decrease in LHRH binding. Much greater inhibition was observed with testosterone and oestradiol. 17-hydroxyprogesterone reduced binding to that of intact controls, whereas dexamethasone was ineffective. When different doses of sex steroids were tested, both oestradiol, testosterone and 5α-dihydrotestosterone inhibited LHRH in a dose-dependent manner. The lowest doses of steroids causing significant inhibition of LHRH binding in castrated animals were 0.5, 50 and 500 μg/day for oestradiol, 5α-dihydrotestosterone and testosterone, respectively. The present study shows that pituitary receptors for LHRH are regulated both by sex steroids and LHRH itself.     

Testicular Tumour Size and Rete Testis Invasion as Prognostic Factors for the Risk of Relapse of Clinical Stage I Seminoma Testis Patients Under Surveillance: a Systematic Review by the Testicular Cancer Guidelines Panel

Context

Patients with clinical stage I (CS I) seminoma testis with large primary tumours and/or rete testis invasion (RTI) might have an increased risk of relapse. In recent years, these risk factors have frequently been employed to decide on adjuvant treatment.

Autoregulation of LH/hCG Receptors and Catabolism of hCG in Rat Testis

The number of free and occupied LH/hCG-receptors in adult rat testes after subcutaneous injections of hCG was studied. When 3–10 IU of hCG was injected, there was initially (between 6 and 16 h) a significant increase in free binding sites, without detectable occupancy of receptors. Higher doses (100 and 1000 IU) induced a drop in the amount of free receptors which was explained by simultaneous receptor occupancy. Later (between 12 and 48 h) there was a pronounced net loss of both free and occupied binding sites. Gel filtration of radioactivity extracted from testicular tissue demonstrated that following the initial uptake [¹²⁵I]hCG undergoes a cleavage to subunits and hydrolysis to peptide fragments and amino acids. The autoradiographic results suggest that the degradative processes take place in Leydig cells. The degradation of [¹²⁵I]hCG was markedly reduced by simultaneous injection of an excess of unlabelled hormone, supporting the autoradiographic evidence of the degradation site of [¹²⁵I]hCG.     

成年大鼠睾丸Smad1和Smad5的免疫组织化学研究

目的 :探讨骨形态形成蛋白的细胞内信号传导分子Smad1和Smad5蛋白在成年大鼠睾丸内的表达。　方法 :应用免疫组织化学ABC法结合葡萄糖氧化酶 DAB 硫酸镍铵增强技术 ,检测成年大鼠睾丸Smad1和Smad5蛋白的表达和定位。　结果 :Smad1蛋白表达于大鼠睾丸精曲小管的各级生精细胞的胞质内 ,呈淡染的紫蓝色颗粒 ,胞核为阴性 ,免疫染色阳性细胞主要包括精原细胞、初级精母细胞、次级精母细胞和精子细胞 ;而Smad5蛋白阳性染色不明显 ,只有睾丸间质细胞呈弱阳性反应 ,免疫反应阳性物质主要定位于细胞质。　结论 :Smad1蛋白主要表达于睾丸精曲小管各级生精细胞 ,Smad5蛋白表达于间质细胞 ,为揭示TGF β超家族在精子发生中的分子机理提供了直接证据。     

年龄相关分子c-kit、HIWI和波形蛋白在不同月龄大鼠睾丸和附睾中的表达

目的：检查c-kit、HIWI和波形蛋白在不同月龄大鼠睾丸和附睾组织中的表达差异,研究睾丸和附睾在成熟和衰老过程中特征性分子表达的规律性改变,为研究男性生殖系统衰老提供实验依据。方法：运用免疫组织化学方法检查6、12、24月龄SD大鼠睾丸和附睾组织中c-kit、HIWI和波形蛋白抗体免疫反应强度,并对其进行比较分析。结果：c-kit特异性免疫反应阳性主要表达于精原细胞,其他细胞表达水平甚弱,不同月龄差异不显著。附睾上皮、附睾管腔内成分亦有c-kit阳性表达。HIWI在大鼠睾丸和附睾组织中表达也很丰富,6月龄和12月龄大鼠中各级生精细胞,尤其是精原细胞和初级精母细胞为免疫反应强阳性细胞;支持细胞、间质细胞、类肌细胞和血管内皮细胞亦为HIWI阳性细胞。附睾上皮有HIWI阳性表达。但是24月龄大鼠睾丸和附睾组织中HIWI表达下调。波形蛋白在睾丸和附睾组织中表达模式为6月龄大鼠睾丸和附睾组织中低表达,24月龄大鼠睾丸和附睾组织表达明显上调（P〈0.01）。结论：c-kit、HIWI在24月龄动物睾丸和附睾组织中表达下调,波形蛋白表达随增龄而明显增强。结果提示,睾丸和附睾组织的衰老与细胞和细胞信号转导异常密切相关。