Seasonal variation in the total volume of Leydig cells in stallions is explained by variation in cell number rather than cell size

Stereological methods were employed in two studies with stallions 1) to determine if seasonal variation in the total volume of Leydig cells is a function of cell number or cell size and 2) to characterize the annual cycle of the Leydig cell population. In the first study, numbers of Leydig cells were calculated for 28 adult (4-20 yr) stallions in the breeding or nonbreeding seasons from nuclear volume density (percentage of the decapsulated testicular volume), parenchymal volume (decapsulated testicular volume), and the volume of individual Leydig cell nuclei. The average volume of the individual Leydig cells was calculated as the total Leydig cell volume/testis (volume density of Leydig cells in the parenchymal volume times parenchymal volume) divided by the number of Leydig cells. The average volume of an individual Leydig cell varied within each season, but means were almost identical for the nonbreeding (6.94 +/- 0.61 picoliter) and breeding (6.91 +/- 0.45 picoliter) seasons. However, Leydig cell numbers per testis were 57% higher in the breeding season, which also had a 58% higher total volume of Leydig cells per testis. In the second study, the numbers of Leydig cells were determined for 43-48 adult horses in each 3-mo period for 12 mo. The number of Leydig cells per testis in May-July was higher (p less than 0.05) than in August-October or February-April, and higher (p less than 0.01) than in November-January. Thus, seasonal fluctuations in the total volume of Leydig cells in adult stallions is a function of the number of Leydig cells that cycle annually.     

Testis morphometry,seminiferous epithelium cycle length,and daily sperm production in domestic cats (Felis catus)

There is very little information regarding the testis structure and function in domestic cats, mainly data related to the cycle of seminiferous epithelium and sperm production. The testis weight in cats investigated in the present study was 1.2 g. Compared with most mammalian species investigated, the value of 0.08% found for testes mass related to the body mass (gonadosomatic index) in cats is very low. The tunica albuginea volume density (%) in these animals was relatively high and comprised about 19% of the testis. Seminiferous tubule and Leydig cell volume density (%) in cats were approximately 90% and 6%, respectively. The mean tubular diameter was 220 microm, and 23 m of seminiferous tubule were found per testis and per gram of testis. The frequencies of the eight stages of the cycle, characterized according to the tubular morphology system, were as follows: stage 1, 24.9%; stage 2, 12.9%; stage 3, 7.7%; stage 4, 17.6%; stage 5, 7.2%; stage 6, 11.9%; stage 7, 6.8%; and stage 8, 11 %. The premeiotic and postmeiotic stage frequency was 46% and 37%, respectively. The duration of each cycle of seminiferous epithelium was 10.4 days and the total duration of spermatogenesis based on 4.5 cycles was 46.8 days. The number of round spermatids for each pachytene primary spermatocytes (meiotic index) was 2.8, meaning that significant cell loss (30%) occurred during the two meiotic divisions. The total number of germ cells and the number of round spermatids per each Sertoli cell nucleolus at stage 1 of the cycle were 9.8 and 5.1, respectively. The Leydig cell volume was approximately 2000 microm3 and the nucleus volume 260 microm3. Both Leydig and Sertoli cell numbers per gram of testis in cats were approximately 30 million. The daily sperm production per gram of testis in cats (efficiency of spermatogenesis) was approximately 16 million. To our knowledge, this is the first investigation to perform a more detailed and comprehensive study of the testis structure and function in domestic cats. Also, this is the first report in the literature showing Sertoli and Leydig cell number per gram of testis and the daily sperm production in any kind of feline species. In this regard, besides providing a background for comparative studies with other fields, the data obtained in the present work might be useful in future studies in which the domestic cat could be utilized as an appropriate receptor model for preservation of genetic stock from rare or endangered wild felines using the germ cell transplantation technique.     

Morphometric studies on hamster testes in gonadally active and inactive states: light microscope findings

This study provides quantitative information on the testes of seasonally breeding golden hamsters during active and regressed states of gonadal activity. Seminiferous tubules occupied 92.5% of testis volume in adult gonadally active animals. Leydig cells constituted 1.4% of the testicular volume. The mean volume of an individual Leydig cell was 1092 microns 3, and each testis contained about 25.4 million Leydig cells. The volume of an average Sertoli cell nucleus during stage VII-VIII of the cycle was 502 microns 3. A gram of hamster testis during the active state of gonadal activity contained 44.5 million Sertoli cells, and the entire testis contained approximately 73.8 million Sertoli cells. Testes of the hamsters exposed to short photoperiods for 12-13 wk displayed a 90% reduction in testis volume that was associated with a decrease in the volume of seminiferous tubules (90.8% reduction), tubular lumena (98.8%), interstitium (72.7%), Leydig cell compartment (79.3%), individual Leydig cells (69.7%), Leydig cell nuclei (50.0%), blood vessels (85.5%), macrophages (68.9%), and Sertoli cell nuclei (34.1%). The diameter (61.1%) and the length (36.8%) of the seminiferous tubules were also decreased. Although the number of Leydig cells per testis was significantly lower (p less than 0.02) after short-photoperiod exposure, the number of Sertoli cells per testis remained unchanged. The individual Sertoli cell in gonadally active hamsters accommodated, on the average, 2.27 pre-leptotene spermatocytes, 2.46 pachytene spermatocytes, and 8.17 round spermatids; the corresponding numbers in the regressed testes were 0.96, 0.20, and 0.04, respectively. The striking differences in the testicular structure between the active and regressed states of gonadal activity follow photoperiod-induced changes in endocrine function and suggest that the golden hamster may be used as a model to study structure-function relationships in the testis.     

Testis morphometry, duration of spermatogenesis, and spermatogenic efficiency in the wild boar (Sus scrofa scrofa)

The wild boar is a natural inhabitant of Europe, Asia, and North Africa and is phylogenetically the ancestor of the domestic pig. Because of its phylogenetic and economic importance, this species is an interesting model for studying testis function in boars. Therefore, the present study was performed to investigate the testis structure, spermatogenic cycle length, and Sertoli cell (SC) and spermatogenic efficiencies in eight adult wild boars. Each spermatogenic cycle lasted 9.05 days, and the total duration of spermatogenesis was estimated as lasting approximately 41 days. The percentages of testis volume occupied by seminiferous tubules and by Leydig cells were 87% and 6%, respectively. The mean number of SCs per gram of testis was 42 million. The SC (round spermatids per SC) and spermatogenic (daily sperm production per gram of testis) efficiencies were 6.6 cells and 28.6 million, respectively. In general, the testis structure, overall germ cell associations at the different stages of the seminiferous epithelium cycle, and duration of spermatogenesis in the wild boar were similar to those in domestic pigs. Probably because of the small size of Leydig cells (400 microm3), their number per gram of testis (157 million) was the highest among investigated mammalian species. Although the SC efficiency in wild boars was low, their spermatogenic efficiency was comparable to that observed in domestic pigs, mainly because of the higher number of SCs per gram of testis in wild boars. These data suggest that SCs became more efficient during evolution, genetic selection, and domestication in pigs.     

Duration of spermatogenesis and daily sperm production in the jaguar (Panthera onca)

The jaguar, like most wild felids, is an endangered species. Since there are few data regarding reproductive biology for this species, our main goal was to investigate basic aspects of the testis and spermatogenesis. Four adult male jaguars were utilized; to determine the duration of spermatogenesis, two animals received an intratesticular injection of H(3)-thymidine. Mean (+/-SEM) testis weight and the gonadosomatic index were 17.7+/-2.2g and 0.05+/-0.01%, respectively, whereas the seminiferous tubules and the Leydig cells volume density were 74.7+/-3.8 and 16.7+/-1.6%. Eight stages of spermatogenesis were characterized, according to the tubular morphology system and acrosome development. Each spermatogenic cycle and the entire spermatogenic process (based on 4.5 cycles) lasted approximately 12.8+/-0.01 and 57.7+/-0.07 d. The number of Sertoli and Leydig cells per gram of testis was 29+/-4x10(6) and 107+/-12x10(6). Based on the number of round spermatids per pachytene spermatocyte (2.8+/-0.3:1; meiotic index); significant cell loss (30%) occurred during the two meiotic divisions. There were approximately eight spermatids for each Sertoli cell (Sertoli cell efficiency), whereas the daily sperm production per gram of testis was 16.9+/-1.2x10(6). We expect that in the near future, the knowledge obtained in the present investigation will facilitate, utilizing germ cell transplantation, preservation of the germinal epithelium and the ability to generate sperm from jaguars in testes of domestic cats.     

Relationships between Leydig cell morphometry and plasma testosterone during postnatal development of the monkey, Macaca fascicularis

In neonates (0 to 3-4 months), the testis contained a mean number of 4.6 X 10(6) Leydig cells representing 4.2 % of its volume; Leydig cell cytoplasm contained 10.2 % of SER. In infants (up to 45 months), Leydig cells regressed but their number increased; their volume density did not change. Leydig cell cytoplasmic volume (454 microns3 ), which was about 2.5-fold less than in neonates (1 119 microns3 ) or adults (1 170 microns3 ), contained only 8.7% of SER. During meiosis stage (38-52 months). Leydig cell numbers and volume density did not vary but the cells reached a maximal size and an amount of SER comparable with that at birth was measured. When spermatogenesis was complete, the Leydig cells represented no more than 0.8% of testis volume, but their number and SER content were significantly increased. Except for a significant decrease when spermatogenesis was completed, Leydig cell lipid content did not change during development, and the volume density of mitochondria did not vary. The mean level of plasma testosterone was 2 ng/ml in neonates and 0.4 ng/ml in infants; it increased to 3 ng/ml during onset of meiosis and reached 10 ng/ml in adults. The profile of testosterone was positively and significantly correlated with the total volume and total number of Leydig cells (P less than 0.01 and P less than 0.02, respectively) and with changes in their cytoplasmic volume (P less than 0.001). Moreover, plasma testosterone levels were positively and significantly correlated with changes in Leydig cell SER content i.e. SER volume density and mean absolute volume per cell (P less than 0.001), total SER in the whole testis (P less than 0.01).     

Comparison of components of the testis interstitium with testosterone secretion in hamster, rat, and guinea pig testes perfused in vitro

Components of the testis and cytoplasmic organelles in Leydig cells were quantified with morphometric techniques in hamster, rat, and guinea pig. Testosterone secretory capacity per gram of testis and per Leydig cell in response to luteinizing hormone (LH) (100 ng/ml) stimulation was determined in these three species from testes perfused in vitro. Numerous correlations were measured among structures, and between structures and testosterone secretion, to provide structural evidence of intratesticular control of Leydig cell function. Testosterone secretion per gm testis and per Leydig cell was significantly different in the three species: highest in the guinea pig, intermediate in the rat, and lowest in the hamster. The volume of seminiferous tubules per gm testis was negatively correlated, and the volumes of interstitium, Leydig cells, and lymphatic space per gm testis were positively correlated with testosterone secretion. No correlations were observed between volumes of blood vessels, elongated spindleshaped cells, or macrophages per gm testes and testosterone secretion. The average volume of a Leydig cell and the volume and surface area of smooth endoplasmic reticulum (SER) and peroxisomes per Leydig cell were positively correlated, and the volume of lysosomes and surface area of inner mitochondrial membrane per Leydig cell were negatively correlated with testosterone secretion. No correlations were observed between volume and surface area of rough endoplasmic reticulum (RER), Golgi apparatus, and lipid, and volume of ribosomes, cytoplasmic matrix, and the nucleus with testosterone secretion per Leydig cell. These results suggest that Leydig cell size is more important than number of Leydig cells in explaining the difference in testosterone-secreting capacity among the three species, and that this increase in average volume of a Leydig cell is associated specifically with increased volume and surface area of SER and peroxisomes. An important unresolved question is what is the role of peroxisomes in Leydig cell steroidogenesis.     

Effect of photoperiod on the size of the Leydig cell population and the rate of recruitment of Leydig cells in adult Syrian hamsters

The number of Leydig cells was determined by stereologic procedures in adult Syrian hamsters housed in long days (14L:10D) to maintain testicular activity (active), in short days (5L:19D) for 12-13 wk to induce testicular regression (photoperiod-induced regressed), or in short days for a period of 21 wk or more to allow spontaneous gonadal recrudescence (spontaneously recrudesced). Testes were removed, sliced, fixed, embedded in Epon 812, and observed by bright-field microscopy. Testicular and seminal vesicle weights, plasma testosterone concentration, total Leydig cell volume per testis, and volume of single Leydig cell were greater (p less than 0.01) in active and recrudesced animals than in regressed animals. The density of Leydig cells was greater in the regressed testes, but the total number per testis was not influenced by photoperiod. In Experiment 2, the rate of recruitment of Leydig cells was determined in 5 adult hamsters exposed to long days (active) or 5 hamsters whose testes were regressed by exposure of animals to short days for 13 wk followed by long-day exposure to initiate testicular growth (photoperiod-induced recrudescing). Hamsters were injected for 3 days/wk for 3 wk with tritiated thymidine, 0.5 or 1 microCi/g body weight. Testes were fixed and tissues prepared, as above, and processed for autoradiography. Again, the photoperiod did not influence the number of Leydig cells per testis. Labeling of Leydig cell nuclei revealed that recruitment of new Leydig cells occurred at approximately 1.3% per day in recrudescing testes but also occurred at approximately 0.6% per day in active testes. Without change in the total number of Leydig cells, new Leydig cells were added continually to the existing population in adult hamsters with either recrudescing or active testes.     

Age-related changes in the function of the pituitary-gonadal axis in a sterile male rat mutant (hd/hd)

Testicular growth is depressed in the genetically sterile male rat (hd/hd) relative to its LE phenotype littermates (by 50% and 73% at 27 and 90 days of age, respectively). Within the hd/hd testis, both the tubular and seminiferous tubule tissues are affected by the mutation. In addition, there is significantly less germ cell production from the primary spermatocyte stage of spermatogenesis onwards and the total number of Sertoli cells observed is less. In the intertubular tissue, the total volume and the total number of Leydig cells per testis is significantly less, but the mean volume of an average Leydig cell is not modified. The serum gonadotropin levels are higher in the hd/hd rat, whereas from 40 days of age onwards the level of testosterone is lower. The FSH and LH binding affinity constants are unchanged by the mutation; however, the total number of FSH binding sites per 10(6) Sertoli cells is lower while that of LH per 10(6) Leydig cells is greater. Indeed, it is likely that the lesser concentration of serum testosterone in the hd/hd rat is a result of a smaller number of Leydig cells since their individual function is not modified. The testicular androgen binding protein (ABP) content and the ABP output towards the epididymis are lower as a consequence of both a lesser number and an altered function of the Sertoli cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Testis stereology, seminiferous epithelium cycle length, and daily sperm production in the ocelot (Leopardus pardalis)

Similar to most wild felids, the ocelot (Leopardus pardalis) is an endangered species. However, knowledge regarding reproductive biology of the ocelot is very limited. Germ cell transplantation is an effective technique for investigating spermatogenesis and stem cell biology in mammals, and the morphologic characterization of germ cells and knowledge of cycle length are potential tools for tracking the development of transplanted germ cells. Our goal was to investigate basic aspects related to testis structure, particularly spermatogenesis, in the ocelot. Four adult males were used. After unilateral orchiectomy, testis samples were routinely prepared for histologic, stereologic, and autoradiographic analyses. Testis weight and the gonadosomatic index were 11 ± 0.6 g and 0.16 ± 0.01%, respectively, whereas the volume density of seminiferous tubules and Leydig cells was 83.2 ± 1.6% and 9.8 ± 1.5%. Based on the acrosomic system, eight stages of spermatogenesis were characterized, and germ cell morphology was very similar to that of domestic cats. Each spermatogenic cycle lasted 12.5 ± 0.4 d, and the entire spermatogenic process lasted 56.3 ± 1.9 d. Individual Leydig cell volume was 2522 μm³, whereas the number of Leydig and Sertoli cells per gram of testis was 38 ± 5 × 10⁶ and 46 ± 3 × 10⁶. Approximately 4.5 spermatids were found per Sertoli cell, whereas daily sperm production per gram of testis was 18.3 ± 1 × 10⁶, slightly higher than values reported for other felids. The knowledge obtained in this study could be very useful to the preservation of the ocelot using domestic cat testes to generate and propagate the ocelot genome.     

Changes in the testis interstitium of Sprague Dawley rats from birth to sexual maturity

Changes in the rat testis interstitium from birth to adulthood were studied using Sprague Dawley rats of 1, 7, 14, 21, 28, 40, 60, and 90 days of age. Our objectives were 1) to understand the fate of the fetal Leydig cells (FLC) in the postnatal rat testis, 2) to determine the volume changes in testicular interstitial components and testicular steroidogenic capacity in vitro with age, 3) to differentially quantify FLC, adult Leydig cells (ALC), and different connective tissue cell types by number and average volume, and 4) to investigate the relationship between mesenchymal and ALC numbers during testicular development. FLC were present in rat testes from birth to 90 days, and they were the only steroidogenic cells in the testis interstitium at Days 1 and 7. Except for FLC, all other interstitial cell numbers and volumes increased from birth to 90 days. The average volume of an FLC and the absolute volume of FLC per testis were similar at all ages except at Day 21, when lower values were observed for both parameters. FLC number per testis remained constant from birth through 90 days. The observations suggested that the significance of FLC in the neonatal-prepubertal rat testis is to produce testosterone to activate the hypothalamo-hypophyseal-testicular axis for the continued development of the male reproductive system. ALC were the abundant Leydig cell type by number and absolute volume per testis from Day 14 onwards. The absolute numbers of ALC and mesenchymal cells per testis increased linearly from birth to 90 days, with a slope ratio of 2:1, respectively, indicating that the rate of production of Leydig cells is 2-fold greater than that of mesenchymal cells in the postnatal rat testis through 90 days. In addition, this study showed that the mesenchymal cells are an active cell population during testis development and that their numbers do not decrease but increase with Leydig cell differentiation and testicular growth up to sexual maturity (90 days).     

Effect of seasonal changes in Leydig cell number on the volume of smooth endoplasmic reticulum in Leydig cells and intratesticular testosterone content in stallions

Testes from 47 adult (4-20 years) stallions obtained in November-January (non-breeding season) and 41 adult stallions obtained in May-July (breeding season) were perfused with glutaraldehyde, placed in osmium and embedded in Epon 812. Percentage Leydig cell cytoplasm or nuclei in the testis was determined by point counting of 0.5 micron sections under bright-field microscopy. Testes from 6 randomly selected horses per season were processed for electron microscopy. The volume (ml) of SER/testis was calculated from the % SER in the cytoplasm % Leydig cell cytoplasm, and parenchymal volume. Number of Leydig cells was calculated from the % nuclei, parenchymal volume, histological correction factor, and volume of single nucleus. Intratesticular testosterone content was determined from the contralateral testis by radioimmunoassay. The volume of SER/g and testosterone/g tended to be higher in the breeding than non-breeding season. Leydig cell number/g, volume of SER/testis, testosterone/testis, and Leydig cell number/testis were significantly greater in the breeding than in the non-breeding season. Volume of SER/testis and testosterone/testis were related significantly to the cell number/testis, and SER/testis was related (P less than 0.05) to testosterone/testis. These results emphasize the importance of seasonal changes in the number of Leydig cells on the amount of SER available to produce testosterone and on testosterone content/testis in the stallion.     

Cell proliferation and hormonal changes during postnatal development of the testis in the pig

Histometrical evaluation of the testis was performed in 36 Piau pigs from birth to 16 mo of age to investigate Sertoli cell, Leydig cell, and germ cell proliferation. In addition, blood samples were taken in seven animals from 1 wk of age to adulthood to measure plasma levels of FSH and testosterone. Sertoli cell proliferation in pigs shows two distinct phases. The first occurs between birth and 1 mo of age, when the number of Sertoli cells per testis increases approximately sixfold. The second occurs between 3 and 4 mo of age, or just before puberty, which occurs between 4 to 5 mo of age, when Sertoli cells almost double their numbers per testis. The periods of Sertoli cell proliferation were concomitant with high FSH plasma levels and prominent elongation in the length of seminiferous cord/tubule per testis. Leydig cell volume increased markedly from birth to 1 mo of age and just before puberty. In general, during the first 5 mo after birth, Leydig cell volume growth showed a similar pattern as that observed for testosterone plasma levels. Also, the proliferation of Leydig cells per testis before puberty showed a pattern similar to that observed for Sertoli cells. However, Leydig cell number per testis increased up to 16 mo of age. Substantial changes in Leydig cell size were also observed after the pubertal period. From birth to 4 mo of age, germ cells proliferated continuously, increasing their number approximately two- to fourfold at each monthly interval. A dramatic increase in germ cells per cross-section of seminiferous tubule was observed from 4 to 5 mo of age; their number per tubule cross-section stabilized after 8 mo. To our knowledge, this is the first longitudinal study reporting the pattern of Sertoli cell, germ cell, and Leydig cell proliferative activity in pigs from birth to adulthood and the first study to correlate these events with plasma levels of FSH and testosterone.     

Developmental stages of fetal-type Leydig cells in prepubertal rats

Fetal Leydig cells were studied in rats during and after the perinatal-neonatal period by comparing changes in morphology, number and volume with changes in testicular steroids and serum luteinizing hormone (LH) concentration. Stereologic examination indicated regression of fetal Leydig cells in testis by showing that their total volume as well as the average cell volume decreased between prenatal day 20 and postnatal day 3. The total number and total volume of cells both increased between postnatal days 3 and 11 but the average cell volume did not change during the same time period. Determination of serum LH showed a close correlation between an increase in LH concentration and increases in total number and volume of cells. The combined number of fetal- and adult-type Leydig cells on day 20 was more than 20 times the number of fetal cells at 3 days of age. Electron microscopic analysis showed that fetal Leydig cells after birth formed conspicuous clusters, which were surrounded by a layer of envelope cells and extracellular material. Occasional dividing fetal Leydig cells and possible precursors of fetal or adult Leydig cells were observed. Mitoses of spindle-shaped pericordal cells were frequent during the neonatal period. During and after the second postnatal week fetal Leydig cells again showed signs of regression, indicated by disintegration of the cell clusters, a decrease in cell size, accumulation of collagen between the cells and a decrease in steroid content per cell.(ABSTRACT TRUNCATED AT 250 WORDS)     

A quantitative study of testicular germ cell populations in masculinized neomale common carp (Cyprinus carpio L.)

The main objective of the present study was to investigate the effects of 17 alpha-methyltestosterone treatment upon the testicular germ cells of gynogenetic masculinized neomale common carp (Cyprinus carpio L.) in comparison with diploid carp. Gynogenetic common carp progeny (mean body weight, BW, 2.6+/-0.3g; mean total length, 10.4+/-0.5 cm) were treated for a period of 40 days with 17 alpha-methyltestosterone (MT) at a dose of 100mg kg(-1). The oral administration of MT resulted in 61.5-100% of fish exhibiting male gonads. The masculinized neomales exhibited reduced (P<0.05) body weight (BW=22.9+/-0.8) but significantly increased (P<0.05) mean testis weight (2.1+/-0.3) and mean gonadosomatic index (GSI=9.5+/-0.2%) in comparison with fish not treated with MT (BW 54.8+/-1.3; GSI=0.61%). Furthermore, treatment with MT also resulted in an increased number of fish exhibiting abnormal gonads. However, neomales did not exhibit abnormalities in the development of sperm ducts. MT treatment significantly increased germ cell volume, nuclear diameter, nuclear volume and cyst volume (P<0.01 in all cases) in MT-treated fish compared to untreated fish. The area occupied by seminiferous tubules, the number of Sertoli cells and germ cells per cyst, and the number of Leydig cells were significantly (P<0.05) greater in fish treated with MT. The carp neomales exhibited approximately 20-60% more Sertoli cells per cyst (P<0.05). Leydig cell nuclear volume and Leydig cell individual volume were significantly reduced in MT-treated groups (P<0.05) compared with untreated groups. In conclusion, our study strongly suggests that the abnormal gonadal structure evident in masculinized neomales could be explained by a combination of MT-induced genetic (homozygosity) and anabolic effects (upon germ and somatic cells).     

Regeneration of Leydig cells in unilaterally cryptorchid rats: evidence for stimulation by local testicular factors

Summary Leydig cells in testes of adult rats were selectively destroyed by a single intraperitoneal injection of ethane dimethane sulphonate. Four days later rats were made unilaterally cryptorchid and 1, 2 and 4 weeks later the histology of the testes was examined by light microscopy and morphometry. After induction of unilateral cryptorchidism, the volume of abdominal compared to scrotal testes was reduced by 45–60% due to rapid impairment of spermatogenesis in abdominal testes. Leydig cells were not present in either scrotal or abdominal testes in the 1-week unilateral crytorchid group. A new generation of foetal-type Leydig cells was observed in scrotal testes of the 2-week unilateral crytorchid group although their total volume per testis estimated by morphometry, was small, being approximately 1 l. In contrast, the abdominal testis exhibited a remarkable proliferation of foetal-type Leydig cells (total volume per testis, 16 l) which predominantly surrounded the peritubular tissues of the seminiferous tubules. A similar morphology and pattern of Leydig cell development was observed in scrotal and abdominal testes of the 4-week unilateral cryptorchid group where total Leydig cell volume was 7 l vs 21 l, respectively. The results show that regeneration of a new population of Leydig cells occurs more rapidly in the abdominal testis than in the scrotal testis of the same animal. These observations suggest the possibility that augmentation of Leydig cell growth is mediated by local intratesticular stimulatory factors within the abdominal testis. Development of new Leydig cells from the peritubular tissue provides circumstantial evidence that the seminiferous tubules and in particular the Sertoli cells, are a likely source of agents that stimulate the growth of Leydig cells.     

The effect of cell damage on the density and steroidogenic capacity of rat testis Leydig cells, using an NADH exclusion test for determination of viability

The ability of cells to exclude pyridine nucleotides was found to be a more sensitive index of cell viability than trypan blue. The three nucleated cell bands obtained by centrifugation of crude rat testis cells on Percoll density gradients contained different proportions of viable and damaged cells. Band III, the steroidogenically active Leydig cell band was mainly viable cells, whilst bands I and II contained 70 and 35% damaged cells respectively. Leydig cells damaged by freezing were shown to have a lower density than viable Leydig cells and did not produce testosterone after stimulation with LH. It is concluded that the methods used to prepare and purify rat testis Leydig cells contribute to the apparent Leydig cell "heterogeneity".     

Down-regulation of steroidogenic cytochrome P450XVII in cryptorchid rat testes

The objective of the present study was to investigate the regulation of a key component of testicular androgen biosynthesis, i.e. the cytochrome P450XVII of the steroid-17 alpha-monooxygenase/C17,20-lyase, after surgical induction of bilateral cryptorchidism in vivo. Seven days after induction of cryptorchidism, P450XVII concentrations are diminished (as compared to sham-operated controls) by 64% in isolated purified Leydig cells but only by 44% in the total Leydig cell compartment of the testis, since the Leydig cell yield from cryptorchid testes is by 53% higher than that from control testes. Using microsomal suspensions prepared from testicular homogenates, P450XVII content per testis equivalent is found to be decreased by 36% seven days after incubation of cryptorchidism, whereas the P450XVII concentration per gram testis is not changed due to testicular involution. Fourteen days after induction of cryptorchidism, the induction of the Leydig cell system appears to superimpose on the down-regulation of P450XVII. The study demonstrates both a strong sensitivity of P450XVII to short-term elevation of testicular temperature and a differentiation between effects of cryptorchidism on total testicular content and specific cellular and subcellular concentration of this steroidogenic protein.     

Signs of aging are apparent in the testis interstitium of Sprague Dawley rats at 6 months of age

The present study investigated the effects of aging in the testis interstitium in Sprague Dawley rats. Rats of 3, 6 and 24 months of age were used. Testes of rats (n = 5) were fixed by whole body perfusion using a fixative containing 2.5% glutaraldehyde in cacodylate buffer, processed and embedded in eponaraldite. Using 1 μm sections stained with methylene blue, qualitative and quantitative morphological studies were performed. Purified Leydig cell preparations, obtained by collagenase digestion followed by elutriation and density gradient centrifugation, were used to determine luteinizing hormone (LH; 100 ng/ml) stimulated testosterone secretory capacity per Leydig cell in vitro. Testosterone levels in the incubation medium, and testosterone and luteinizing hormone levels in serum of these three groups of rats were determined via radioimmunoassay. Morphological studies revealed that Leydig cells were more abundant in the testis interstitium at 6 and 24 months when compared to 3 months. Moreover, collagen fiber bundles were more frequently observed in the testis interstitium at older ages. Blood vessels of the testis interstitium in 24-month-old rats frequently showed partial and complete occlusion of their lumen and thickening of vessel walls. This feature was also present at 6 months, but less frequently. The results of the sterological studies revealed that the volumes of seminiferous tubules, interstitium and Leydig cells per testis was significantly higher (P < 0.05), at 6 and 24 months of age than those at 3 months. Moreover, volume of macrophages per testis was observed to be significantly higher (P < 0.05) at 6 months when compared to 3 and 24 months, and volume of connective tissue cells per testis was observed to be significantly lower (P < 0.05) at 6 and 24 months when compared to 3 months of age. No significant difference (P > 0.05) was observed for the volume of lymphatic space per testis in the three age groups studied. Volume of interstitial blood vessels per testis was not significantly different at 3 and 6 months of age, but a significantly greater (P < 0.05) volume was observed at 24 months. However, at 6 and 24 months, only 71% and 31% of the total blood vessel volumes respectively had completely open lumen in them; the rest of the blood vessels were either partially (12.5% at 6 months and 17% at 24 months) or completely (16.5% at 6 months and 52% at 24 months) occluded. The number of Leydig cells per testis was doubled at 6 and 24 months of age compared to 3 months. The average volume of a Leydig cell was not significantly different between 3 and 6 months of age, however, at 24 months a significantly lower (P < 0.05) value was observed. LH stimulated testosterone secretory capacity per Leydig cell in vitro was reduced by 50% at 6 months of age compared to 3 months; a further significant (P < 0.05) reduction was observed at 24 months. Serum testosterone and LH levels were not significantly different between 3 and 6 months of age but at 24 months a significantly lower (P < 0.05) value was observed for both of these hormones.In summary, the present study demonstrated many changes in the components of the testis interstitium in the aged Sprague Dawley rat. Modifications in the blood vessels and the occurrence of abundant collagen fibers in the interstitial space could possibly contribute to the reduced testosterone secretory capacity per Leydig cell with advancing in age. The observed Leydig cell hyperplasia could be suggested as a compensatory effort to maintain the normal androgen status of the aged rat, which is rather successful at 6 months but unsuccessful at 24 months. This investigation further revealed that these characteristic changes in the aged testis interstitium at 24 months are also present to some extent at 6 months of age in Sprague Dawley rats, suggesting that aging of the testis in this strain of rats commences early in life.     

Exposure to constant light during testis development increases daily sperm production in adult Wistar rats.

Testis histometry and daily sperm production (DSP) were evaluated in adult (160-day-old) Wistar rats exposed to constant light for the first 25 days after birth, and compared with control animals which were exposed to a 12 h-light-12 h-dark light regimen. Significantly greater (P < 0.05) numbers of Sertoli cell nucleoli and round spermatids per cross-section of seminiferous tubule were found in animals exposed to constant light. In addition, epididymis weight, DSP per testis and per gram of testis, as well as Leydig cell compartment volume, were significantly increased in treated animals. Although there was a clear trend toward an increased Sertoli cell population per testis in animals exposed to constant light, this difference was not statistically significant (P < 0.05). The number of round spermatids as expressed per Sertoli cell was the same in both groups. Surprisingly, the diameter and volume of round spermatid nucleus at stages I and VII of the cycle of seminiferous epithelium were significantly lower (P < 0.05) in treated animals. In conclusion, constant illumination during neonatal testis development increased sperm production and Leydig cell compartment volume in adult rats probably through a mechanism involving elevated follicle stimulating hormone and luteinizing hormone during the prepubertal period. To our knowledge, this is the first study showing that altering the light regimen can affect sperm production in non-seasonal breeders.