Experimental cryptorchidism in the adult mouse: I. Qualitative and quantitative light microscopic morphology

Morphologic changes in the testes of adult mice after experimentally induced cryptorchidism were studied by light microscopy and stereology. Increasing duration of cryptorchidism resulted in a gradual decrease in the volume of seminiferous tubules per testis, and this was associated with germ cell degeneration. The volumes of Sertoli cell lipid droplets increased, and dilations of the intercellular space between the Sertoli cell junctions was observed in the cryptorchid testis. The luminal volume of the seminiferous tubule was reduced by 50% after 28 days of cryptorchidism. However, the volumes of intertubular tissue and Leydig cells in control and cryptorchid testes were not significantly different. Leydig cell number per testis increased, and the average volume of a Leydig cell decreased gradually with the progression of the cryptorchid state. The volume of the connective tissue cells in the intertubular area increased, but no significant volume change was observed in the volume of intertubular macrophages. After 28 days, the cryptorchid testis contained a significantly increased volume of blood vessels and a reduced volume of lymphatic space per testis. These observations clearly demonstrate that, although the mouse is a species closely related to the rat, the morphologic changes that occur in the Leydig cell population after induction of experimental cryptorchidism in this species is different.     

Leydig cell cytoplasmic content is related to daily sperm production in men

The relationship between the abundance of specific Leydig cell organelles and daily sperm production (DSP) was determined. Testes from 10 men (26-53 years of age) were obtained at autopsy within 10 h of traumatic death or heart failure and fixed by vascular perfusion. Testicular tissue was processed for light and electron microscopy. DSP/testis and Leydig cell cytoplasmic volume/testis were determined by stereology of histologic sections. The Leydig cell organelle content was determined by point counting electron micrographs for smooth endoplasmic reticulum (SER), rough endoplasmic reticulum, mitochondria, lipofuscin pigment, lipid, Golgi bodies, and Reinke crystals. Men were divided equally into two groups based on DSP/testis. Men with low DSP/testis had less SER volume density (P less than 0.01) and lower SER volume per testis (P less than 0.05) than men with high DSP. Other organelles were unrelated to DSP. When all men were combined, the volume density of SER (r = 0.80; P less than 0.01), the volume SER per testis (r = 0.69; P less than 0.05), and the volume SER per Leydig cell (r = 0.84; P less than 0.01) were significantly related to DSP. Hence, there appears to be a significant relationship between Leydig cell SER and the level of spermatogenesis in men.     

Effect of unilateral cryptorchidism on the intertubular tissue of the adult rat testis: evidence for intracellular changes within the Leydig cells

Adult male rats were made unilaterally cryptorchid for 1, 2 or 4 weeks, and the morphological response of the Leydig cells was then studied using morphometric assessment of total Leydig cell volume and number per testis in abdominal and scrotal testes. Serum hormone levels were measured and the steroidogenic properties of isolated Leydig cells were evaluated by in-vitro stimulation with hCG and interstitial fluid (IF) obtained from normal rat testes. Total Leydig cell volume and number per testis were not altered in abdominal vs scrotal testes, although the volume of the abdominal testis was 46, 29 and 21%, respectively, of the volume of the contralateral scrotal testis after 1, 2 and 4 weeks. This reduction was accompanied by significant (P less than 0.05) elevation of the serum levels of FSH and LH, although serum testosterone levels were unchanged from the normal range. Despite the lack of quantitative alterations in Leydig cell morphology, hCG- and IF-stimulated testosterone production was significantly (P less than 0.01) greater by abdominal Leydig cells when compared with scrotal Leydig cells derived from the same animals. Ultrastructural examination of Leydig cells in situ suggested an increase in volumetric density of mitochondria in abdominal Leydig cells. Together with the enhanced steroidogenic responses of these cells, these findings suggest that disruption of spermatogenesis in the cryptorchid testis is accompanied by intracellular activation of Leydig cells. Since these effects were not exhibited by Leydig cells from the scrotal testis it is concluded that local factors within the cryptorchid testis are responsible, at least in part, for regulation of Leydig cell activity.     

Fine structure of the Sertoli cells of the rat testis in experimental unilateral cryptorchidism

Mature rats were made unilaterally cryptorchid for 28 days. ABP concentrations of the abdominal epididymis were significantly reduced, indicating impaired secretory activity of the Sertoli cells. The FSH serum level was increased and testosterone levels both of the serum and of homogenates of the cryptorchid testis were slightly reduced. Sertoli cells of the cryptorchid testis showed signs of regression. Although Sertoli cell junctions were still impermeable to lanthanum, a reduction of membrane particles forming the Sertoli cell junctions was observed in freeze-fracture replicas. In thin sections it was seen that the plasma membrane-apposed cisternae of endoplasmic reticulum or the adjaacent microfilaments were partially lacking. No such changes were observed in the scrotal testis.     

Fine Structure of the Sertoli Cells of the Rat Testis in Experimental Unilateral Cryptorchidism

Mature rats were made unilaterally cryptorchid for 28 days. ABP concentrations of the abdominal epididymis were significantly reduced, indicating impaired secretory activity of the Sertoli cells.
The FSH serum level was increased and testosterone levels both of the serum and of homogenates of the cryptorchid testis were slightly reduced. Sertoli cells of the cryptorchid testis showed signs of regression. Although Sertoli cell junctions were still impermeable to lanthanum, a reduction of membrane particles forming the Sertoli cell junctions was observed in freeze-fracture replicas. In thin sections it was seen that the plasma membrane-apposed cisternae of endoplasmic reticulum or the adjaacent microfilaments were partially lacking. No such changes were observed in the scrotal testis.     

Morphometric analysis of the components of the neonatal and the adult rat testis interstitium

Leydig cells in the foetal rat testis are still present at birth and it has been hypothesized that they commence to degenerate immediately after birth, based on the decrease in their volume density (v/v%) with age. In this study the interstitium of the rat testis was studied quantitatively at 1, 5, 10, 15, 20 and 90 days after birth: the latter are considered to be adults. The absolute volumes of connective tissue cells and blood vessels increased with age. The absolute volumes of macrophages and lymphatic spaces were greater at 90 days than at any other age. The absolute volume of foetal Leydig cells per testis was unchanged from 1 to 15 days, despite a decrease in the % volume occupied per testis. The number of foetal Leydig cells per testis did not decline from days 1-20 although on day 20 an average foetal Leydig cell was smaller in volume than at earlier ages (days 1-15). Adult Leydig cells were recognized at day 10 and their absolute volume and number per testis increased from 15 to 90 days. Adult Leydig cells were similar in morphology to foetal Leydig cells at 20 days except for a reduced volume of cytoplasmic lipid.     

Postpubertal cryptorchism. Morphofunctional study with special reference to Leydig's cells

In a group of 17 patients of postpubertal age with unilateral (n = 15) or bilateral (n = 2) cryptorchism, a significant decrease in the tubular diameter was observed, in addition to Leydig cell hyperplasia (many with cytoplasm vacuolization and/or atrophy) in both the cryptorchid testes and in the contralateral scrotal testes. The number of testosterone-positive Leydig cells in testicular tissue sections, studied with peroxidase-antiperoxidase, was diminished in the cryptorchid testes, whereas in the contralateral scrotal testes it was similar to the control group. Together with normal testosterone levels and elevated luteinizing hormone and follicle-stimulating hormone levels in peripheral blood, this leads us to think of a compensated dysfunction of the Leydig cells. This possible lower testosterone production by the Leydig cells in the cryptorchid testis is not borne out morphologically, where the volume of the organelles is similar to the contralateral scrotal testes.     

Effect of cryptorchidism on testicular and Leydig cell androgen production in the mouse

Unilateral cryptorchidism was induced surgically in adult mice and the effects on testicular and Leydig cell steroidogenesis were studied after 7 weeks. There was a 60% reduction in weight of the cryptorchid testis and this was associated with a significant reduction in intratesticular androgen content, both under basal conditions and following an injection of hCG. Testicular androgen production in vitro was also significantly lower in the cryptorchid testis compared to the scrotal testis, again under both basal conditions (29 +/- 6% of control) and in the presence of hCG (46 +/- 9% of control). Scrotal testes from the unilaterally cryptorchid animals did not show any significant difference in steroidogenic capacity compared to testes from untreated control animals. The decrease in steroidogenic capacity of the cryptorchid testis was due, at least in part, to a reduction in activity for each Leydig cell. In four experiments, androgen production by Leydig cells isolated from cryptorchid testes was 48 +/- 9% of cells from scrotal testes in the presence of a saturating dose of hCG. Under basal conditions the effect was more variable between experiments with steroid secretion by Leydig cells from cryptorchid testes being 58 +/- 32% of that for cells from scrotal testes. Leydig cell steroidogenesis in the scrotal testes of unilaterally cryptorchid animals did not differ significantly from untreated controls. These results show that induced cryptorchidism in the mouse causes a significant reduction in Leydig cell activity. This is apparently different from the effects of this procedure on the rat and raises the possibility that intratesticular regulation differs between the two species.     

Effect of neonatal treatment of rats with potent or weak (environmental) oestrogens,or with a GnRH antagonist,on Leydig cell development and function through puberty into adulthood

This study addressed whether reduced Sertoli cell number or manipulation of the neonatal hormone environment has an influence on final Leydig cell number per testis in the rat, by applying neonatal treatments known to affect these parameters, namely administration of a GnRH antagonist (GnRHa) or diethylstilboestrol (DES, in doses of 10, 1 or 0.1 microg per injection). The effect of treatment with either of two 'environmental oestrogens', bisphenol-A (Bis-A) or octylphenol (OP), was also evaluated. Leydig (3beta-hydroxysteroid dehydrogenase immunopositive) cell development and function (plasma testosterone levels) were studied through puberty into adulthood. Treatment with GnRHa impaired testis growth, Leydig cell (nuclear) volume per testis and testosterone levels during puberty, when compared with controls, but final Leydig cell volume/number in adulthood was comparable with controls. As adult testis weight was reduced by 45% in GnRHa-treated rats, the percentage Leydig cell volume per testis was approximately double (p < 0.01) that in controls, and also at day 35. Testosterone levels in adulthood in GnRHa-treated rats were lower (p < 0.01) than in controls but were within the lower end of the normal range. Treatment with DES caused largely dose-dependent suppression of testis growth, Leydig cell (nuclear) volume per testis and testosterone levels up to day 35. Although by adulthood, Leydig cell volume/number per testis was comparable with controls in DES-treated rats, testosterone levels remained grossly subnormal. Neonatal treatment with either Bis-A or OP had little consistent effect on any of the parameters studied except that both treatments significantly elevated testosterone levels on day 18, as did treatment with DES-0.1 microg. The present findings are interpreted in the context of what is known about the hormonal regulation of Leydig cell development. These lead to the conclusion that final Leydig cell number per testis is not determined by the number of Sertoli cells per testis and appears not to be influenced in any major way by gonadotrophins, androgens or oestrogens in the first 2 weeks of postnatal life. This implies that adult Leydig cell number may be determined prior to birth.     

Early signs of Sertoli and Leydig cell dysfunction in the abdominal testes of immature unilaterally cryptorchid rats

Testicular descent was prevented unilaterally by cutting the gubernaculum testis of newborn rats. When 20 days old unilaterally cryptorchid rats were injected intraperitoneally with 2 μg bFSH per gram body weight and killed 6 h later when testicular testosterone (T) and oestradiol (E₂) concentrations were determined. The increase in E₂ was subnormal in abdominal testes. In 18-day-old unilaterally cryptorchid rats the efferent ducts were ligated bilaterally, and the rats were killed 48 h later. The weight increase, due to accumulation of seminiferous tubule fluid, was significantly greater in the abdominal testes. In contrast, the ABP content of the abdominal epididymis was subnormal in 20-day-old unilaterally cryptorchid rats. Unilateral orchidectomy was performed in 16-day-old unilaterally cryptorchid rats and at 20 days of age intratesticular T and E₂, and plasma FSH and LH concentrations were determined and compared to that in 20-day-old control unilaterally cryptorchid rats. Removal of an abdominal testis resulted in increased plasma FSH and intratesticular E₂, whereas plasma levels of LH and intratesticular levels of T were unaffected. Removal of a scrotal testis resulted in increased plasma FSH and LH coupled with increased intratesticular T and E₂. Rats with a single abdominal testis had higher plasma FSH and LH and intratesticular T, but similar intratesticular E₂, than rats with a single scrotal testis. It is concluded that Sertoli and Leydig cell function are influenced by cryptorchidism at a stage when the temperature difference, and the morphological differences between the testes are very discrete.     

Experimental cryptorchidism in the adult mouse: II. A hormonal study

Serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone levels and secretory capacity of the in vitro stimulated testis were determined in control and bilateral cryptorchid mice after 7, 14, 21, and 28 days. In a separate study, serum FSH, LH, and testosterone levels were measured in unilateral and bilateral cryptorchid, hemicastrate, and bilaterally castrate adult mice after 28 days of treatment. Serum FSH levels were significantly increased in bilaterally cryptorchid mice compared to controls (0 days) at 7, 14, 21, and 28 days, but serum LH and testosterone levels did not change. At 28 days, the elevated serum FSH levels in the unilateral and bilateral cryptorchid mice were not different than those in hemicastrate mice. However, the FSH levels in bilaterally castrate mice after 28 days were significantly higher than all other groups. Serum LH and testosterone levels were significantly different only in the bilaterally castrate group, compared to control levels. Both the normal and cryptorchid testes of all ages studied were capable of producing equal levels of testosterone in vitro, both basally and with a human chorionic gonadotropin (hCG) dose of 700 mIU/ml (maximum stimulatory dose for both the normal and the 28 day cryptorchid testes). Changes that occur in mice with experimentally induced cryptorchidism are not identical to those seen in the rat. Serum FSH levels increase, but no changes occur in serum LH and testosterone levels. Additionally, a cryptorchid mouse testis is not hyperresponsive to hCG stimulation in vitro.     

双侧短期隐睾大鼠睾丸形态及抑制素的研究

本文研究了双侧短期人为隐睾大鼠睾丸的形态及抑制素的变化。结果发现隐睾一周和四周的大鼠睾丸和付睾重量均减轻。曲细精管直径缩小(平均直径:对照组250μm±43.75μm,1周组,159.5±32.25;4周组139.25±22.5;与对照组比P均<0.01)。Leydig细胞切面面积比明显增大(对照组为(％)6±2.4,1周组为21.83±7.4,4周组为23±11.38,P均<0.01)。电镜发现隐睾组leydig氏细胞线粒体和内浆网数量均增多,局部呈囊性扩张。睾丸间液和血清抑制素含量均明显下降(与对照组比P分別<0.01和<0.05),表明曲细精管破坏与leydig细胞呈一定的负相关。支持精管控制leydig氏细胞的理论,是否抑制素参于这一过程有待证实。     

Effects of hypophysectomy and alterations in spermatogenic function on Leydig cell volume, number, and proliferation in adult rats

The two major objectives of this study were to determine (i) whether the pituitary is required to maintain Leydig cell number per testis, and (ii) whether alterations in spermatogenic function in the absence of LH can affect Leydig cell volume, number, and 3H-thymidine incorporation in vivo. Four experimental treatments tested the combinations of two factors: (i) the intact pituitary vs hypophysectomy (Hypox); and (ii) arrested vs active spermatogenesis. Subdermal Silastic capsules were used to deliver a low dosage of estradiol in addition to a low dosage of testosterone (TE), which arrested spermatogenesis, or a high dosage of testosterone (HTE) which maintained active spermatogenesis. All four treatments (TE, HTE, Hypox and Hypox-HTE) inhibited LH secretion for 16 weeks. Control rats were sham hypophysectomized. Leydig cell volume per testis and the volume of an average Leydig cell decreased 70-85% (P less than 0.01 vs controls) in all treated rats, whether deprived of LH (TE) or of all pituitary secretions (Hypox), and whether spermatogenesis was arrested (TE, Hypox) or maintained by exogenous testosterone (HTE, Hypox-HTE). This result suggested that LH was the only factor required to maintain Leydig cell volume, since the absence of other pituitary factors or alterations in spermatogenic function could not override or modify the effect of LH deprivation. No significant differences were found in Leydig cell number per testis or the proportion of Leydig cells labeled with 3H-thymidine among control and experimentally treated rats. In contrast to Leydig cell volume, which depended on LH, Leydig cell number and Leydig cell division were maintained at control values in the absence of pituitary factors and spermatogenic function for 16 weeks.     

Mitosis in normal adult guinea pig Leydig cells

In this study, Leydig cells in mitosis in adult guinea pigs were quantified. Testes of adult control guinea pigs (n = 10) were fixed by whole body perfusion with 2.5% glutaraldehyde in cacodylate buffer, postfixed in a mixture of osmium tetroxide-potassium ferrocyanide, and embedded in Epon Araldite for qualitative and quantitative microscopy. Using stereologic techniques, the total number of Leydig cells per testis and the number of dividing Leydig cells per testis were quantified. Light microscopic studies revealed the presence of dividing Leydig cells. Ultrastructural studies on a few of these Leydig cells showed that they contained abundant smooth endoplasmic reticulum and mitochondria, which further supported this identity. The total number of Leydig cells per testis and the number of dividing Leydig cells per testis were determined as 14.1 x 10(6) (standard error [SE] = 0.33) and 8 x 10(3) (SE = 5), respectively. These results indicate that Leydig cells undergo mitosis at a rate of 1 per 1.75 x 10(4) in adult guinea pigs.     

Morphological and Endocrinological Differences between the Abdominal Testes in Bilateral and Unilateral Cryptorchid Rats

Using a new experimental model of cryptorchism in rats, where testicular descent was prevented, testicular development and function were studied in bilateral and unilateral cryptorchid animals Morphometric and radioimmunological techniques were used. Up to 30 days after birth testicular development was identical in the two types of abdominal testes but in adult rats differences were observed. In these rats spermatogenesis was damaged to a similar extent, but total tubular length and testicular weight were increased in the bilateral abdominal testes. Moreover, in these testes, the volume density of Leydig cells, the total Leydig cell mass, the average Leydig cell size and the testis testosterone concentration were larger than in unilateral abdominal testes. It is suggested that the impaired spermatogenesis seen in both kinds of abdominal testes may be unrelated to Leydig cell function.     

The meaning of the Leydig cell in relation to the etiology of cryptorchidism: An experimental electron-microscopic study.

In our electron-microscopic studies of testicular biopsies, both normal and cryptorchid, we found a simple atrophy of the Leydig cell in the cryptorchid testis. Based on experiments by Raynaud1,2 and Jean3 on pregnant mice, we tried to find the reason for changes in the Leydig cell relating to the etiology of cryptorchidism. We found on electron microscopic study of testes in the offspring of pregnant mice treated with estrogen the same atrophy of the Leydig cell as we see in human cryptorchidism. These changes are not evident when estrogen and HCG are given together. We can conclude from this experiment that lack of gonadotropin stimulation leads to the atrophy of Leydig cells. This atrophy then produces a lack of androgen which could be responsible for cryptorchidism.     

Ultrastructural interrelationship between the pineal gland and the testis in the male rat

The ultrastructural interrelationship between the pineal gland and testis was evaluated in the rat. Wistar rats were divided into 6 groups. Groups I and II were sham-orchidectomized and orchidectomized rats, respectively. Rats in group III were orchidectomized and daily injected with testosterone propionate (TP) for 1 month. Groups IV and V were sham-pinealectomized and pinealectomized, respectively. Group VI was pinealectomized and daily injected with melatonin for 2 months. All animals were anesthetized with ketamine for fixation by vascular perfusion. Pineal glands of groups I, II, and III and the testes of groups IV, V, and VI were removed and weighed. All specimens were examined by electron microscopy. Orchidectomy caused an increase of lipid droplets, cytoplasmic dense bodies, and lysosomes. Rough endoplasmic reticulum, Golgi apparatus, and mitochondria were extensive in the cytoplasm. TP administration to orchidectomized rats resulted in formation of less extensive lipid droplets and mitochondria. In pinealectomized rats, golgi complex, mitochondria, and enlarged smooth endoplasmic reticulum were extensive in the cytoplasm of Leydig cells. Formation of cytoplasmic secretory granules and osmiophilic bodies was observed. Testicular weight increased compared to group IV. Melatonin decreased testicular weight in comparison to group V and prevented ultrastructural changes. Pinealectomy and orchidectomy caused hyperactivity in Leydig cells and pinealocytes, respectively, which suggests a mutual relationship between the pineal gland and testis in the rat.     

Morphometric and stereological study of the glandular epithelium of the lateral prostate of the intact and castrated guinea pig

The glandular epithelium of the lateral prostate of the guinea pig was described within the framework of a morphometric model in terms of relative densities and absolute dimensions. A combination of direct measurement and point and intersection counting techniques was used. The quantitative data generated in the intact animals were compared with those of castrated controls. Castration was accompanied by a significant decrease in height of the glandular epithelium and in sizes of secretory and basal cells and their corresponding nuclei. On a per cell basis, significant decreases in total volume and surface area of granular endoplasmic reticulum were detected after castration. This was accompanied by a significant reduction in the total volume of Golgi cisternae. The total volume, surface area, and number of highly electron-dense and clear granules decreased significantly compared with the intact control animals. However, no significant changes in these parameters of low electron-dense granules were found. Significant reductions in the total volume and surface area of condensing granules, lysosomes, and mitochondria, but not their number, were detected. The average sizes of condensing granules, secretory granules, lysosomes, and mitochondria were decreased significantly after castration. The present study showed that the alterations in the secretory function of the secretory cells of the lateral prostate was reflected by the quantitative changes in granular endoplasmic reticulum, Golgi complexes, and secretory granules on a per cell basis. The data generated in the present study will serve as a baseline for further studies of the lateral prostate of the guinea pig.     

Morphometric and stereological analysis of the effects of 17 beta-estradiol on the glandular epithelium of the castrated guinea pig lateral prostate.

Upon administration of pharmacological doses of estradiol to castrated guinea pigs, the secretory cells of the lateral prostate underwent hypertrophy which resulted from significant increases in nuclear and cytoplasmic volume. There were quantitative increases in the small highly electron-dense granules and multivesicular bodies when compared with the castrated control. The dramatic increase in the number of highly electron-dense granules probably occurred at the expense of the low electron-dense granules. The average size of the condensing granules and mitochondria decreased significantly after estradiol administration. However, significant increase in the number of mitochondria was detected when compared with the castrated control. Ultrastructural data revealed no significant changes in the absolute dimensions of granular endoplasmic reticulum or of the Golgi complex, suggesting that estradiol exerted no significant stimulatory effects on these organelles. Pharmacological doses of estrogen appear to regulate the expression of secretory granules and multivesicular bodies in the lateral prostate of castrated guinea pigs.     

Leydig cell hyperplasia in cryptorchid patients: Quantitative evaluation of Leydig cells in undescended and contralateral scrotal testes

Summary Leydig cell number was evaluated quantitatively in testicular biopsies from post-pubertal cryptorchid patients and normal controls. For this quantitative evaluation we used the following method. This is based on the determination of the total number of Leydig cells, Leydig cell clusters and seminiferous tubules in the entire histologic sections of each biopsy and the determination of the following indices; mean Leydig cells per tubule, mean Leydig cell clusters per tubule and mean Leydig cells per cluster. In addition, the numbers of Sertoli cells were counted, and Leydig-Sertoli cell ratio was also determined. These indices were correlated with each other. All indices were significantly elevated not only in undescended but in contralateral scrotal testes of the cryptorchid patients in comparison to those in normal controls. Between undescended and descended scrotal testes of the same individual patients, those indices were significantly higher in the descended scrotal testes than in the undescended ones. Thus, Leydig cell hyperplasia was noted in the testes of post-pubertal cryptorchid patients, and was more prominent in the contralateral scrotal testes than in the undescended ones.