Changes in daily sperm production rate in rats under the influence of a potent antispermatogenic agent, CDRI 84/35

CDRI 84/35, a potent nonsteroidal antispermatogenic agent, causes total sterility in rats by directly acting on germ cells while having no effect on Sertoli/Leydig cells. This study was conducted to evaluate the effect of the compound on gametogenic activity of testes and to identify stages of spermatogenesis that were affected. Adult male rats administered either compound 84/35 at minimum effective dose or estradiol (5 μg) or water only were killed on days 22, 41, and 64 of the treatment period to evaluate the effect on spermatid, spermatocyte, and spermatogonial stages, respectively. Daily sperm production (DSP) was measured employing a homogenization technique. Results showed a decline in testis weight and DSP with a drastic reduction (95%) in DSP in 84/35-treated rats on day 41 of the treatment period. Estradiol was more potent in reducing the testis weight; however, 84/35 had an edge over estradiol in reducing the DSP. After withdrawal of treatment for 120 days, a phenomenal recovery (>90%) in DSP per gram parenchyma was noted in 84/35-treated animals. Results indicate a direct effect of estradiol on spermatogonia, whereas 84/35 seems to affect the spermatocyte stage.     

Effect of antispermatogenic agents on cell marker enzymes of rat Sertoli cells in vitro

OBJECTIVE: To examine the role of Sertoli cells in the antispermatogenic action of two nonsteroidal male contraceptive compounds (CDRI-84/35 and gossypol) by evaluating their effect on some key parameters of Sertoli cell function in vitro. METHODS: Primary cultures of Sertoli cell were established from 18-day-old rat testis and treated on day 5 with different concentrations (1.0, 0.1, 0.01, and 0.001 mM) of either CDRI-84/35 or gossypol in vitro. Lactate (secretion), along with beta-glucuronidase, gamma-glutamyl transpeptidase, lactate dehydrogenase (LDH) and aromatase activities, was measured in these cells to examine the functions targeted by antispermatogenic agents in Sertoli cells. RESULTS: CDRI-84/35 significantly affected Sertoli cell parameters (stimulation in most of the cases) that are important for germ cell development like lactate secretion, LDH activity, aromatase activity (estradiol secretion) and so on. Gossypol in comparison to CDRI-84/35 had a more severe effect on Sertoli cells with complete inhibition of enzyme activities at higher concentrations. CONCLUSION: It is probable that the antispermatogenic action of CDRI-84/35 and gossypol is routed through Sertoli cells by disruption of important cell functions that support spermatogenesis in vivo. However, the two compounds appear to have different course of action in Sertoli cells, ultimately leading to spermatogenic failure.     

Reversible antispermatogenic effect of gossypol in langur monkeys (Presbytis entellus entellus)

The present investigation reports the antispermatogenic effect of the orally active highly purified gossypol acetic acid at 7.5 mg and 10 mg/day for 180 days in langur monkeys. The results revealed a dose-dependent response in semen analysis as well as testicular morphology. Uniform severe oligospermia was observed in the lower dose (7.5 mg) group, while azoospermia was observed in 2 out of 5 animals in the higher dose (10 mg) group and the remaining animals showed severe oligospermia. Scanning electron microscopy of spermatozoa revealed deleterious abnormalities in the head and midpiece. Testicular morphology revealed a decrease in the seminiferous tubule diameter and arrest of spermatogenesis. The lower dose group had a germ cell population up to primary spermatocytes while the higher dose group had only Sertoli cells and spermatogonia. Withdrawal of treatment for 180 days led to the recovery of all the parameters studied, to normalcy.     

Recent studies on lonidamine,the lead compound of the antispermatogenic indazol-carboxylic acids

Lonidamine (LND) or [1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic acid] is an anticancer and an antispermatogenic drug whose mechanism of action is still incompletely understood. LND is effective against a number of tumors, including head, neck and breast cancers, probably because of the inhibition of mitochondrial electron transport and the enzyme hexokinase and to the induction of apoptosis. Instead, the antispermatogenic activity of LND appeared to be related not only to its energolytic activity but also to other effects activities such as the inhibition of specific chloride channels in the epididymis and the disruption of the inter-Sertoli-germ cell junctions, leading to premature release of germ cells. In addition, we recently reported that, in the rat, LND at the dose of 100 mg/Kg b.w. p.o., a fully active but well tolerated dose, caused specific changes of the testicular and epididymal macroglobulins (alpha(2)-macroglobulin, alpha(1) inhibitor-3 and alpha(1)-macroglobulin). Further studies are needed to elucidate the mechanism of action of LND, the lead compound of an interesting class of antispermatogenic drugs based on the core structure of 1-(2,4-dichlorobenzyl)-indazole-3-carboxylic acid.     

Long-term effects of lonidamine on mouse testes

Lonidamine (LND) [1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic acid] is a well-known antispermatogenic drug. The aim of this study was to identify its possible long-term sequelae on the reproductive system of mice as compared with rats, where most data have been obtained until now. Sexually mature CD1 male mice were administered a single dose of LND (200 mg/kg bw by gavage) and killed 24 and 48 h, 6 days and 2, 4 and 8 weeks after the treatment. Testes were collected, weighed and (1) fixed in Bouin's solution for histological analysis or (2) reduced to monocellular suspensions and ethanol fixed to undergo flow cytometry (FCM) DNA content analysis. No effect on body weight and/or food consumption was observed in the treated group in comparison with the control group. Testicular weight was significantly reduced 24 h after the treatment. Reduced seminiferous epithelium with a progressive lack of intercellular cohesion and marked depletion of spermatids, infiltration of granulocytes, desquamation into the tubular lumen and increased intertubular spaces were present by 24 h after the treatment and persisted to a marked degree at 48 h, 6 days and 2 and 4 weeks up to a marked degeneration of tubular structures with absence of spermatogenesis. The same effects, albeit with a moderate severity, were still present 8 weeks after the treatment. As also detected by FCM, primary spermatocytes appeared to be the main cellular target. Sertoli and Leydig cells were remarkably spared. The histological findings are consistent with those previously observed in rats and point out that testicular damage may persist for several weeks after a single-dose administration. Findings are discussed in comparison with testicular toxicity elicited by other xenobiotics.     

Lonidamine transiently affects spermatogenesis in pubertal CD1 mice

Lonidamine (LND) [1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic acid], a well-known antispermatogenic drug, was studied for the first time in pubertal mice to assess its possible effects on spermatogenesis. Male CD1 mice were orally treated on Postnatal Day (PND) 28 with a single dose of LND (100 mg/kg body weight) and sacrificed on PND30, PND42, PND74 and PND123. On PND30 (48 h after dosing), severe testicular effects were evidenced in the treated animals: (a) reduction of the testicular sperm head concentration (approximately 50% of the control value); (b) changes in the spermatogenic cell type distribution (mild decrease of the elongated spermatids and S-phase cells fractions); and (c) morphological alterations of the Sertoli cell cytoplasm and germ cell exfoliation. These changes were recovered in adulthood, on PND74 and PND123. However, no effect on sperm chromatin structure was detected on the epididymal sperm of mature mice by sperm chromatin structure assay, suggesting that LND did not interfere with the process of chromatin reorganization and DNA packaging.     

THYROID HORMONES: THEIR ROLE IN TESTICULAR STEROIDOGENESIS

Thyroid hormones are important for growth and development of many tissues. Altered thyroid hormone status causes testicular abnormalities. For instance, juvenile hypothyroidism/neonatal transient hypothyroidism induces macroorchidism, increases testicular cell number (Sertoli, Leydig, and germ cells) and daily sperm production. Triiodothyronine (T3) receptors have been identified in sperm, developing germ cells, Sertoli, Leydig, and peritubular cells. T3 stimulates Sertoli cell lactate secretion as well as mRNA expression of inhibin- &#102, androgen receptor, IGF-I, and IGFBP-4. It also inhibits Sertoli cell mRNA expression of Müllerian inhibiting substance (MIS), aromatase, estradiol receptor, and androgen binding protein (ABP) and ABP secretion. T3 directly increases Leydig cell LH receptor numbers and mRNA levels of steroidogenic enzymes and steroidogenic acute regulatory protein. It stimulates basal and LH-induced secretion of progesterone, testosterone, and estradiol by Leydig cells. Steroidogenic factor-1 acts as a mediator for T3-induced Leydig cell steroidogenesis. Although the role of T3 on sperm, germ, and peritubular cells has not yet been completely studied, it is clear that T3 directly regulates Sertoli and Leydig cell functions. Further studies are required to elucidate the direct effect of T3 on sperm, germ, and peritubular cells.     

Intrauterine bisphenol A exposure leads to stimulatory effects on Sertoli cell number in rats

Using the optical disector for quantifying cell numbers, we investigated whether oral treatment of rats on days 6-21 of gestation with the weakly estrogenic bisphenol A (BPA, 0.1 or 50 mg/kg) or the highly estrogenic ethinyl estradiol (EE, 0.02 mg/kg) alters testicular histology, in those offspring 9-12 month of age. Since production of male germ cells depends on Sertoli cell number, possible changes in that parameter were investigated using unbiased stereology. Spermatogenesis was qualitatively normal in all groups. BPA increases Sertoli cell number per organ but not when expressed as per gram testis. EE did not affect cell number per organ but did affect numbers on a per gram testis basis due to a lowered testis weight. In contrast to the lowering of Sertoli cell numbers that might have been expected according to the estrogen hypothesis, intrauterine administration of these xenoestrogens in fact resulted in minor increases in Sertoli cell numbers and had no qualitative effect on spermatogenesis.     

Quinalphos-induced suppression of spermatogenesis, plasma gonadotrophins, testicular testosterone production, and secretion in adult rats.

Quinalphos (O,O-diethyl-O-[quinoxalinyl-(2)-thionophosphate]) is a well-known organophosphorus insecticide used extensively in agriculture that adversely interferes with the activity of testicular steroidogenic enzymes in rats. To investigate its effects on spermatogenesis, the other function of testes, quantitative evaluation of different varieties of germ cells at stage VII of the seminiferous epithelium cycle, namely, type A spermatogonia (ASg), preleptotene spermatocytes (pLSc), midpachytene spermatcytes (mPSc), and step 7 spermatids (7Sd), along with the radioimmunoassay of plasma FSH, LH, testosterone, and testicular testosterone, were performed in Wistar rats following treatment with quinalphos (250 micrograms/kg, ip) for approximately one (13 days) and two cycles (26 days) of the seminiferous epithilium. Massive degeneration of all varieties of germ cells at stage VII, remarkable reduction in the sperm count, and significant reductions in plasma concentrations of FSH and testosterone, along with testicular testosterone, were observed after quinalphos treatment. Significant reduction in the plasma concentration of LH was observed only after treatment for two cycles. Administration of human chorionic gonadotrophin for 26 days in rats injected with quinalphos partially prevented the degeneration of germ cells and increased testosterone production. It is suggested that quinalphos may have a suppressive influence on gonadotrophin release but its direct detrimental action at the level of the testes may also be responsible for the observed changes in spermatogenesis and in testicular testosterone production in rats.     

双酚A雄性生殖毒性的体内外实验研究

目的探讨双酚A对雄性动物生殖机能的影响。方法将双酚A混入饲料(0、1和5g／kg)连续饲喂成年32只SD大鼠14d,放免法测定睾酮和雌二醇并进行右睾组织形态分析;对原代培养的支持细胞染毒(0、10^-7、10^-6、10^-5、10^-4moL／L)。结果5g／kg的双酚A组的右睾平均重量1．53g显著低于对照组1．62g,但1g／kg组与对照组差异无统计学意义。形态观察发现,2个双酚A组中的曲细精管基底膜均与生精细胞分离;部分生精细胞和支持细胞发生核固缩和空泡变性;同时,双酚A处理使黏附于支持细胞的生精细胞平均数量由对照组的7．94个分别减少为4．13和3．04个。此外,双酚A在体内外实验中均抑制睾丸支持细胞的波形蛋白表达,阻碍细胞骨架和胞间联结的形成,使支持细胞在体外培养中的形态变得异常细长。但双酚A对血清雌二醇和睾酮浓度的影响没有统计学意义。结论双酚A可能通过破坏支持细胞骨架和改变支持细胞形态而损害雄性生殖功能。     

Effect of high dose of pyridoxine on mammary tumorigenesis

The effect of high-dose pyridoxine (PN) on mammary tumorigenesis was examined in female Sprague-Dawley rats. The first mammary tumors appeared between 84 and 90 days after 7,12-dimethylbenzanthracene treatment. There was no effect of PN level on tumor incidence at 90 days but at 98, 104, and 111 days. Tumor incidence was lower in the high-dose group (35 mg PN/kg diet) compared with the controls (7 mg PN/kg diet). All tumors were identified as adenocarcinoma and most as papillary type. The number of microcarcinomas in mammary glands of the 35-mg PN group tended to be reduce than that of the 7-mg group. The number of proliferating Ki67-positive cells was significantly reduced by supplementation with PN.     

用睾丸细胞共培养探讨吡哆醇对大鼠睾丸细胞的毒性

目的探讨吡哆醇（ＰＮ）对大鼠睾丸的体外毒性。方法采用在Ｗｉｌｉａｍｓ方法的基础上改进的Ｓｅｒｔｏｌｉｇｅｒｍ细胞共培养系统,观察ＰＮ在不同剂量和接触时间对培养细胞的作用。结果脱落生精细胞数随ＰＮ浓度的增高和接触时间的延长而增加,并有明显的剂量—效应和时间—效应关系。同时,还观察到Ｓｅｒｔｏｌｉ细胞骨架出现松弛、回缩等效应。结论ＰＮ对大鼠生精细胞的体外效应反映了其对Ｓｅｒｔｏｌｉ细胞的损害。睾丸细胞共培养方法对探讨ＰＮ对大鼠睾丸的毒性作用具有实用价值。     

雷公藤糖浆对人精子发生影响的研究

对服用雷公藤糖浆患者的69份精液标本和2例睾丸活检组织进行了光镜和透射电镜形态学观察。结果显示:服药15日以上者,其精液中可见大量脱落的精子细胞和精母细胞,这些细胞均有明显的超微病理变化。皋丸曲细精管中精子细胞和精母细胞脱落至净,支持细胞的紧密连接有超微病理变化,但是,精原细胞未见形态学异常。提示雷公藤的“毒性”物质可能主要作用于睾丸曲细精管连腔小室内的生殖细胞。该结果为服用雷公藤可影响精子发生,停用雷公藤后精液常规能恢复正常提供了形态学依据。     

Antispermatogenic effects of tolnidamine in langur (Presbytis entellus)

Tolnidamine (50 mg/kg body weight; twice a week; oral) was administered for 90 days to adult male langur monkeys (Presbytis entellus entellus Dufresne) to assess its contraceptive potential. Semen weight, volume, seminal fluid volume, colour, pH and libido remained unchanged. Sperm motility, vitality and morphology were impaired with the advancement of treatment. Sperm density reduced to severe oligospermia following 75-90 days of treatment. Increased number of immature germ cells were also noticed. Resumption of changes to pretreatment range was observed following 90 days of cessation of treatment. However, sperm density remained low all through the recovery period of 150 days. Seminal fructose, ACP, LDH and citric acid concentrations did not change markedly. A significant depletion in GPC and magnesium levels was recorded during treatment and early recovery periods. Alterations in germ cells and Sertoli cells were also observed. A progressive but reversible rise in serum creatinine was evident. Other clinical parameters and body weight response revealed no drug-related alterations. In conclusion, tolnidamine medication induced irreversible inhibition of spermatogenesis.     

大剂量吡哆醇的睾丸毒性体内外效应的比较研究

目的：　探讨大剂量吡哆醇（ＰＮ）的大鼠睾丸的毒性及可能机制。方法：采用腹腔注射和Ｓｅｒｔｏｌｉ－ｇｅｒｍ 细胞共培养两种模式,观察睾丸及副性腺变化。结果：注射１５ 天３００ｍ ｇ 和６００ｍ ｇ 组睾丸及副性腺萎缩,光镜和电镜下见精子细胞脱落、精母细胞变性坏死、精子释放延迟和异形精子,Ｓｅｒｔｏｌｉ细胞肿胀、微管稀少,内质网异位和扩张等。注射３０ 天效应更为明显。体外实验显示ＰＮ剂量与生精细胞脱落显著相关,并与Ｓｅｒｔｏｌｉ细胞骨架的变化一致。结论：　吡哆醇可能主要通过损伤Ｓｅｒｔｏｌｉ细胞,引起睾丸结构和功能改变。     

Alteration in testicular cell components following transiently induced ischaemia in prepubertal bulls

The aim of the present study was to evaluate transient testicular ischaemia (induced using elastrator bands) in Jersey calves on testicular morphology and development. Treatments (at 27 +/- 5 days of age) consisted of control (0 h banding) and banding for 2, 4 or 8 h (n = 4 in each group). After castration (at 60 +/- 5 days of age), the right testis was used for calculation of cell components per testis according to the point-counting method. Bodyweight (59.8 +/- 6.2 kg) and scrotal circumference (SC) at banding (9.1 +/- 0.2 cm) did not differ between groups. Fresh testis weight, scrotal temperature immediately before band removal and daily SC growth were decreased in ischaemic (4 and 8 h) testes compared with controls (P < 0.05). In addition, the number of Sertoli and Leydig cells was significantly reduced in the 8 h ischaemic treatment group (P < 0.05). Transiently induced ischaemia significantly decreased the number of germ cells in the 8 h ischaemic treatment group (13 +/- 5 x 10(6) cells) compared with the 0, 2 and 4 h ischaemic treatment groups (38 +/- 6, 32 +/- 6 and 33 +/- 5 x 10(6) cells, respectively; P < 0.05). These results suggest that transiently induced ischaemia for 8 h significantly decreases the number of germ, Sertoli and Leydig cells in prepubertal testis.     

Sites of action of gossypol studied by autoradiography and enzyme histochemistry

The distribution of 14C-gossypol acetate was studied by autoradiography in male rats after intraperitoneal or intratesticular injection. Accumulation of radioactivity was found in testis, kidney and liver, while there was little in brain, pituitary and epididymis. In testis, high accumulation occurred in interstitial cells, with low levels in Sertoli cells, spermatogonia and spermatocytes. In addition, the chronic effect of gossypol was assessed by enzyme histochemistry with thiamine pyrophosphate, alpha-glycerophosphate dehydrogenase, and by lipid stain. In the treated animals an increased number of luminal exfoliated cells (Sertoli cells, germ cells and spermatids) was noted, which showed positive reactions. The results suggest both direct and indirect effects of gossypol on testicular functions.     

超生理剂量11-酸睾酮对人精液中脱落生殖细胞的影响

为研究大剂量睾酮对生精细胞的影响,对53例志愿接受11-酸睾酮的成年男性在给药前后及恢复期的精液进行分析,用瑞-姬氏染色进行脱落生殖细胞观察。结果为给药后30,60天脱落生殖细胞计数随精子计数减少而减少,初级精母细胞、次级精母细胞比例增多,精子细胞减少。细胞增多了凋亡脱落生殖细胞的各种形态变化,包括凋亡的初级、次级精母细胞和精子细胞。恢复期随精子计数的恢复,生精细胞的计数和形态也相应恢复。提示11-酸睾酮主要干扰了初级、次级精母细胞和精子细胞的正常分化发育。