Effect of clove flower extract (Syzygium aromaticum) on spermatogenic cells and Leydig cells in the unilateral cryptorchidism albino rat model (Rattus norvegicus)

This study aimed to determine the effect of the administration of ethanolic extract of Moringa oleifera leaves on the number of spermatogenic and Leydig cells of gentamicin-induced rats (Rattus norvegicus). This study used 25 white male rats divided randomly into five groups. The rats were injected with Gentamicin 5 mg/kg bw/day subcutaneously for 14 days, except for the negative control group (NCG). During the next 14 days, the rats were given oral moringa leaf extract of 200, 316, and 500 mg/kg bw/day respectively for E200, E316, and E500 groups. The data were analyzed with one-way ANOVA followed by Duncan's test. The results showed that the number of spermatogonia, spermatocytes, and spermatids in NCG was higher (p <0.05) than in the other groups. The number of spermatogonia, spermatocytes, and spermatids in the E316 group was higher (p <0.05) than in PCG. The number of spermatogonia in the E500 group was higher (p <0.05) than in the E316 group, but the number of spermatocytes and spermatids in the E316 group was similar (p >0.05) to the E500 group. The number of PCG Leydig cells was the smallest (p <0.05) compared to the other groups. The administration of Moringa leaves extract in the E200 and E316 groups increased (p <0.05) the number of Leydig cells compared to PCG. The number of Leydig PCG cells in the E500 group was not significantly different (p >0.05) from that of the NCG. It could be concluded that administration of Moringa oleifera leaves extract restored the number of spermatogenic and Leydig cells of gentamicin-induced rats.

This study aimed to determine the effect of the administration of ethanolic extract of Moringa oleifera leaves on the number of spermatogenic and Leydig cells of gentamicin-induced rats (Rattus norvegicus). This study used 25 white male rats divided randomly into five groups. The rats were injected with Gentamicin 5 mg/kg bw/day subcutaneously for 14 days, except for the negative control group (NCG). During the next 14 days, the rats were given oral moringa leaf extract of 200, 316, and 500 mg/kg bw/day respectively for E200, E316, and E500 groups. The data were analyzed with one-way ANOVA followed by Duncan's test. The results showed that the number of spermatogonia, spermatocytes, and spermatids in NCG was higher (p <0.05) than in the other groups. The number of spermatogonia, spermatocytes, and spermatids in the E316 group was higher (p <0.05) than in PCG. The number of spermatogonia in the E500 group was higher (p <0.05) than in the E316 group, but the number of spermatocytes and spermatids in the E316 group was similar (p >0.05) to the E500 group. The number of PCG Leydig cells was the smallest (p <0.05) compared to the other groups. The administration of Moringa leaves extract in the E200 and E316 groups increased (p <0.05) the number of Leydig cells compared to PCG. The number of Leydig PCG cells in the E500 group was not significantly different (p >0.05) from that of the NCG. It could be concluded that administration of Moringa oleifera leaves extract restored the number of spermatogenic and Leydig cells of gentamicin-induced rats.

Aim:Biotechnological culture of hypoxia-conditioned (CH) rat mesenchymal stem cells (rMSC-CH) for testicular failure therapy with low libido improves the functional outcome of the testicle for producing spermatogenic cells and repairs Leydig cells in rats (Rattus norvegicus).Materials and Methods:In the first group (T1), rats with testicular failure and low libido were injected with normoxia-conditioned (CN) rMSCs (21% oxygen); in the second group (T2), rats with testicular failure and low libido were injected with rMSC-CH (1% oxygen); in the negative control group (T−), rats with normal testis were injected with 0.1 mL phosphate-buffered saline (PBS); and in the sham group (TS), rats with testicular failure and low libido were injected with 0.1 mL of PBS.Results:Vascular endothelial growth factor expression, as the homing signal, in the groups T2, T−, T1, and TS was 2.00±0.5%, 2.95±0.4%, 0.33±0.48%, and 0±0%, respectively. The number of cluster of differentiation (CD)34+ and CD45+ cells in the groups T− and TS was <20%, whereas that in T1 and T2 groups was >30% and >80%, respectively, showing the mobilization of hematopoietic stem cells (HSCs). The number of spermatogenic cells (spermatogonia, primary spermatocytes, secondary spermatocytes, and spermatid) decreased significantly (p<0.05) in TS compared with that in T−, T1, and T2, whereas that in T2 did not show a significant (p>0.05) decrease compared to that in T−. The improvement in libido, based on the number of Leydig cells producing the hormone testosterone for libido expression, did not increase in T1, whereas T2 was able to maintain the number of Leydig cells significantly compared to that between TS and T1.Conclusion:rMSC-CH culture for testicular failure with low libido showed improvement in the functional outcome of the testicle and in repairing Leydig cells.

The effects of pure FSH and/or LH preparations on the number of Leydig cells and their function in immature hypophysectomized rats have been investigated. As a result of hypophysectomy at the age of 17-18 days, the number of recognizable Leydig cells per testis decreased, as did the steroidogenic capacity in vivo and in vitro. Treatment with 64 micrograms FSH on both 22 and 23 days of age, did not affect the number of recognizable Leydig cells. In contrast, two injections of LH (10 micrograms) caused a sixfold increase in the number of Leydig cells, but had a negative effect on spermatogenesis. These stimulatory and inhibitory effects of LH diminished when FSH was added. Treatment with FSH for 7 days caused a twofold increase in the number of Leydig cells when compared with hypophysectomized controls. 3 beta-Hydroxysteroid dehydrogenase (3 beta-HSD) and esterase activity in Leydig cells also increased under the influence of FSH. The pregnenolone production per Leydig cell in the presence of 5-cholesten-3 beta,22(R)-diol (22R-hydroxycholesterol) as substrate showed a sevenfold increase. Plasma testosterone levels 2 h after injection of human chorionic gonadotrophin in intact rats and hypophysectomized FSH-treated rats were the same. Following LH treatment for 7 days, the number of Leydig cells proved to be 11 times higher, and 3 beta-HSD and esterase activity were not different from intact controls. The testicular pregnenolone production was four- to fivefold higher when compared with untreated hypophysectomized rats. However, pregnenolone production per Leydig cell in LH-treated rats was only slightly different from the hypophysectomized controls.(ABSTRACT TRUNCATED AT 250 WORDS)

This research aimed to determine the effect of α-tocopherol on the count of spermatogenic and Leydig cells in rats (Rattus norvegicus) exposed to 2,3,7,8 tetrachlorodibenzo-p-dioxin (dioxin). Dioxin is an endocrine-disrupting agent that adversely affects reproductive health, while α-tocopherol maintains fertility. This research used 25 rats aged 10-12 weeks weighing 150-200 grams. Rats were divided into five groups (K, P0, P1, P2 and P3). The K (control group) was administered with corn oil 1 ml/day. P0 was exposed to 700 ng/kg/day dioxin. P1, P2 and P3 was exposed to dioxin at a dose of 700 ng/kg/day and administered with α-tocopherol at a dose of 77, 140 and 259 mg/kg/day respectively. Dioxin exposure, corn oil and α-tocopherol administration were conducted orally for 20 days. On day-21, all rats were sacrificed for histological slides preparation of testicles with hematoxylin-eosin staining. Data were analyzed with analysis of variance and continued with the Duncan test. The results indicated that exposure to dioxin caused a decrease in the number of spermatogenic and Leydig cells. The administration of α-tocopherol at a dose of 140 mg/kg/day eliminated the effect of reducing the number of spermatogenic and Leydig cells caused by exposure to dioxin. The conclusion was the administration of α-tocopherol at 140 mg/kg/day was effective in maintaining the number of spermatogenic and Leydig cells in rats (Rattus norvegicus) exposed to 2,3,7,8 tetrachlorodibenzo-p-dioxin.

High temperatures lead to oxidative stress, which can disturb spermatogenesis process. Watermelon (Citrullus lanatus) peel contain antioxidant expected to compensate oxidative stress due to heat stress exposure. This study aimed to determine the effect of watermelon rind ethanolic extract on the number of Leydig, Sertoli, and spermatogenic cells of rats exposed to heat (40°C). Twenty rats (Rattus norvegicus) were divided randomly into five groups. In the control group (T0) rats were not exposed to heat nor given the watermelon rind extract. T1, T2, T3, and T4 groups were exposed to heat for an hour daily and orally given placebo (1% Na CMC), 100, 200, and 400 mg/kg BW of watermelon rind extract (in 1% Na CMC). Rats were treated for 52 days, and sacrificed for the testicle collection. Hematoxylin-eosin stained histological slides were prepared for the examination of Leydig, Sertoli and spermatogenic cells. The results showed no significant difference (p &gt;0.05) in the average number of Leydig cells in rats among groups. The number of Sertoli cells and spermatogenic cells of rats exposed to heat (T1) was lower than those of the normal rats (T0 group). The dose of watermelon rind ethanolic extract at 200 mg/kg BW (T3 group) and 400 mg/kg BW (T4) increased (p &lt;0.05) the number of Sertoli and spermatogenic cells. It could be concluded that a dose of 400mg/kg BW of watermelon rind ethanolic extract maintained the number of Leydig cells, Sertoli cells, and spermatogenic cells of rats exposed to heat.

We have previously demonstrated that neonatal and transient neonatal hypothyroidism modulates Leydig, Sertoli, and germ cell numbers, sex steroids and androgen binding protein concentration. The present study was undertaken to study the effect of neonatal onset hypothyroidism on Leydig and peritubular myoid cell numbers, plasma and testicular interstitial fluid (TIF) sex steroid concentration at different age groups of Wistar rats. Hypothyroidism was induced by giving 0.05% methimazole (MMI) to lactating mothers or directly to the male pups from day 1 postpartum through days 10, 15, 30, 40 and 60 postpartum. To confirm hypothyroidism, plasma thyroid hormones and TSH were assayed. Plasma and TIF testosterone, progesterone, dihydrotestosterone (DHT) and estradiol were assayed by radioimmunoassay. Leydig cell number in hypothyroid rats were less than the age-matched controls. The diameter of Leydig cells in hypothyroid rats was smaller than the controls but 10 days old hypothyroids alone had larger than control rats. A significant decrease of peritubular myoid cell number was observed in 30, 40 and 60 days hypothyroid rats and increased in 10 and 15 days hypothyroidism. Hypothyroid rats had elevated level of plasma LH and decreased GH (except day 10 postpartum). Plasma PRL level was increased in 10 and 15 days hypothyroid rats and an opposite effect was observed in 40 and 60 days hypothyroidism. Plasma testosterone, DHT and estradiol were decreased in all hypothyroid rats. However, plasma progesterone level in hypothyroid rats was significantly higher at days 10, 30, and 40 postpartum and an opposite effect was seen in 15 and 60 days experimental groups. TIF testosterone and progesterone titre showed a consistent decrease in hypothyroid rats irrespective of the duration. In hypothyroid rats, TIF DHT levels decreased significantly in days 10, 40 and 60 postpartum. However, it increased in days 15 and 30 postpartum. Except at day 10 postpartum, the level of TIF estradiol in hypothyroid rats was significantly less than their age matched controls. Our data clearly indicate that neonatal onset hypothyroidism adversely affect Leydig cell proliferation along with impaired steroidogenesis.

Macrophages are a common cell type in the testicular interstitium of the rat and are morphologically and functionally related to Leydig cells. We investigated the number of macrophages and Leydig cells in long-term (24 weeks) hypophysectomized (LTHX) or sham-operated rats. LTHX rats showed a 76% decrease in the number of macrophages, whereas the number of Leydig cells was only slightly decreased (by 18%). The profile areas of both macrophages and Leydig cells were very much decreased (46% and 66% respectively). Sham-operated and LTHX rats were treated with vehicle or human FSH and LH (hFSH/hLH; 75 IU/kg body weight per day) for 1 week. This treatment induced a 286% increase in the number of macrophages and a 32% increase in the number of Leydig cells in LTHX rats. The profile areas of macrophages and Leydig cells were also increased (212% and 184% respectively). About 80% of macrophages showed vacuolization of the cytoplasm. Gonadotrophin treatment did not induce changes in cell numbers in sham-operated animals but about 30% of macrophages showed large cytoplasmic vacuoles. Vehicle- or hormone-treated LTHX rats were given a single injection of ethylene dimethane sulphonate (EDS) and killed 72 h later. Leydig cells were absent from the testicular interstitium of sham-operated rats but there were large numbers of dead Leydig cells (about 40% of the pre-existing population) in the testicular interstitium of LTHX rats 3 days after EDS treatment. Complete clearance of the testicular interstitium from EDS-killed Leydig cells was found in LTHX rats treated with hFSH/hLH.(ABSTRACT TRUNCATED AT 250 WORDS)

Diethylstilbestrol induces morphological changes in the spermatogonia, Sertoli cells and Leydig cells of adult rat

Histomorphological and histochemical features of testes were studied in six adult indigenous bulls (Bos indicus) of two different age groups, 1 year 9 months to 2 years of age (group A) and 2 years 3 months to 2 years 6 months of age (group B) during the period from September 2006 to April 2007 by using Hematoxylin and Eosin (H&amp;E) stain, Verhoeff's stain, Van Gieson's stain and Periodic Acid-Schiff Reaction (PAS) stain. The testes were surrounded by visceral layer of tunica vaginalis (consisted of mesothelium and connective tissue) and tunica albugenia mainly composed of collagen fibers. The seminiferous tubules were tortuous, two ended loops and varying in appearance and the wall of tubules consisted of lamina propria, basement membrane supported by reticular fibers and a lining of complex stratified epithelium consisted of sertoli cells and spermatogenic cells. The sertoli cells are irregulary columnar cells, extended from basal lamina to lumen of tubules and the spermatogenic cells situated between the sertoli cells in an orderly manner with four to eight layers occupying the space between the basal lamina and the lumen of the tubules. There was presence of both spermatid and spermatozoa in the lumen of some seminiferous tubules of testes of bulls of both age groups. The spermatogonia, primary spermatocytes and secondary spermatocytes showed more staining affinity than the spermatid in routine staining technique. The basement membrane of tubules, spermatid and spermatozoa showed positive affinity whereas spermatogonia, primary spermatocytes and secondary spermatocytes showed negative affinity to PAS stain. The interstitial tissues located between the sminiferous tubules, consisted of connective tissue network, mainly composed of collagenous and reticular fibers; blood and lymph vessels with Leydig cells. The Leydig cells were present as single or groups within intertubular spaces. It was concluded that the thickness of tunica albuginea, the stratification of growing spermatogenic cells and cross sectional length and breadth of the seminiferous tubules of testes were higher in the bull of group B than group A and the number of Leydig cells were more in the testis of group A than group B and in between left and right testes, the thickness of tunica albuginea and cross sectional length and breadth of the seminiferous tubules were higher in the left testis but the number of Leydig cells was higher in right testis in both age groups. Key words: Testis, seminiferous tubule, Leydig cell, indigenous bull DOI = 10.3329/bjvm.v6i1.1341 Bangl. J. Vet. Med. (2008). 6 (1): 67-74

The Leydig cells of young hypophysectomized rats are highly sensitive to the stimulatory effects of exogenous pituitary hormones. The aim of this study was to analyse the role of testicular macrophages in the response of Leydig cells to different hormones. Male rats were hypophysectomized at 28 days of age and 10 days later they were injected intratesticularly with dichloromethylene diphosphonate-containing liposomes (right testis) to deplete testicular macrophages, and with 0.9% NaCl (left testis). One week later, the animals were treated daily with 1 IU rat GH (rGH)/rat, 5 IU recombinant human FSH (recFSH)/rat, 10 IU human chorionic gonadotrophin (hCG)/rat, or vehicle for 7 days. The animals were killed on the day after the last injection. The animals treated with rGH showed increased body weight and increased number and size of testicular macrophages in the left testes, but no significant effects on Leydig cells were found. Treatment with recFSH induced a significant increase in testicular weight and tubular diameter in both testes. In the left testes, the number and size of macrophages were increased; the number of Leydig cells was not changed, although they showed a significantly increased cross-sectional area. This effect was abolished in the right (macrophage-depleted) testes. However, the effect of recFSH on the growth of the seminiferous tubules was not modified by the absence of macrophages. Rats treated with hCG showed increased testicular weight and serum testosterone levels, as well as an increased weight of the ventral prostate. In the left testes, the number and size of both macrophages and Leydig cells were increased. Otherwise, the number of Leydig cells was unchanged in the absence of macrophages, whereas the increase in the size of Leydig cells was partially abolished. These data indicate that testicular macrophages are needed for the response of Leydig cells to gonadotrophin treatment.

BACKGROUND Chronic stress increases glucocorticoid levels and accelerates reduction in Leydig cells functions and numbers. Chronic stress models in the working place comprise sleep deprivation, sedentary stress, and physical stress. The aim of this study was to evaluate the effect of various work stress models, such as stress from paradoxical sleep deprivation (PSD), immobilization, and footshock, on serum testosterone levels and number of Leydig cells in male albino rats. METHODS This study was of experimental randomized post-test only with control group design using 24 male Wistar albino rats (Rattus norvegicus). The sample was divided into 4 groups: K1 (control), K2 (PSD), K3 (immobilization) and K4 (footshock), receiving treatment for 25 days. Measured parameters were serum testosterone level and Leydig cell number. Analysis of variance (ANOVA) was used for statistical analysis, followed by post hoc LSD. RESULTS Mean serum testosterone levels (0.07 ± 0.08 ng/ml) and Leydig cell numbers (4.22 ± l0.96) were lowest in the PSD stress model. Serum testosterone levels differed significantly between controls and PSD group (p=0.014), while there was a significant difference in numbers of Leydig cells between footshock stress and PSD (p=0.011) and between the three stress groups and controls (p=0.006). CONCLUSION This study demonstrated that PSD, immobilization and footshock stress significantly decreased serum testosterone levels and number of Leydig cells in male albino rats (Rattus norvegicus). The mechanism by which PSD affects serum testosterone is still unclear.

In an effort to further understand the basis for the changes in steroidogenesis known to occur during sexual maturation in the rat, we examined by quantitative morphologic methods the number and ultrastructure of Leydig cells in fetal rats (days 18-20 of gestation) and in rats from days 2 to 3 of age through adult. Quantitative light microscopic analyses indicated that Leydig cell number, when expressed per unit volume of testis, was very high in fetal rat testes, fell significantly in testes of days 2 to 3 rats, and subsequently rose significantly. When Leydig cell number was expressed per testis rather than per unit volume of testis, the results indicated that testes of fetal rats and rats of days 2 to 3 contained the same number of Leydig cells; after the neonatal period, significant increases in Leydig cell number per testis occurred in concert with increases in testis weight. Quantitative electron microscopic studies revealed significant differences in the ultrastructure of fetal and adult populations of Leydig cells. For example, Leydig cells of fetal and neonatal rats contained abundant lipid, whereas Leydig cells of weeks 7 to 8 and adult rats contained little. Stereological analyses also revealed dramatic changes in smooth endoplasmic reticulum and inner mitochondrial membrane surface areas during sexual maturation, both per cell and per testis. These findings are discussed with respect to the steroidogenic capacity of the testis during sexual maturation.

The effect of chronic luteinizing hormone treatment on adult rat Leydig cells

We have previously observed (M. O. Suescun et al., 1994, Journal of Andrology, 15, 442-448) that rats with autoimmune orchitis (EAO) exhibit increased testosterone production in vitro by isolated testes. The aim of the present study was to determine whether the increase in testosterone production correlated with an enhanced number of Leydig cells and/or enhanced steroidogenic capacity per Leydig cell. For this purpose, EAO was induced in adult Sprague-Dawley rats by active immunization with testicular homogenate and adjuvants. At 80 days after the primary immunization, 60% of rats presented with severe testicular damage characterized by sloughing of the seminiferous epithelium, seminiferous tubule atrophy and interstitial mononuclear cell infiltration. At 160 days after the first immunization, testicular lesions were more severe. A morphometric study, by light microscopy, showed an increase in the number of Leydig cells in rats with EAO (45% increase at 80 days and 50% at 160 days). By electronmicroscopy, testicular sections of rats with EAO revealed the presence of numerous Leydig cells closely associated with macrophages. Most Leydig cells exhibited ultrastructural features of active steroid secreting cells. The steroidogenic capacity of Percoll-purified Leydig cells from tests of rats with EAO, killed at 80 and 160 days, was evaluated. Leydig cells from rats with EAO exhibited an enhanced steroidogenic response to hCG in vitro at 80 days (38%) and an increase in basal (77%) and post-hCG testosterone production (115%) at 160 days compared to controls. However, these cells were less sensitive to hCG. In conclusion, the results indicate that the enhancement of in-vitro testosterone production observed in rats with EAO is accounted for both by the increased number of Leydig cells and by the increased testosterone production of each Leydig cell.