Spermatogenic Cells In Seminiferous Tubules Research Articles

The effects of saline water consumption on sperm parameters, testicular histopathology, hormonal and antioxidants concentrations in Barki Rams

The study aimed to assess the effects of water salinity on the sperm parameters, levels of cortisol, LH, FSH, testosterone and antioxidants as well as the testes’ histopathology in Barki rams. Fifteen healthy Barki rams (1–1.5 years) were divided into three equal depending on the type of drinking water for nine months. The rams in the tap water group (TW, water that contained 350 ppm of total dissolved salts (TDS). Males in the high saline water group (HSW) were permitted to consume high saline water with 8,934 ppm TDS, whereas those in the second group were permitted to have moderately saline water (MSW, 4,557 ppm TDS). High salt concentration in drinking water had adverse effect on sperm viability, morphology and sperm cell concertation. Nitric oxide and malondialdehyde concentrations in blood were significantly higher in the MSW and HSW groups than in TW. There was a significant decrease in glutathione concentration as well as superoxide dismutase activity in TDS and HSW. Cortisol was most highly concentrated in the HSW, next in the MSW, and least in TW. The testosterone, LH, and FSH concentrations in the HSW and MSW groups were significantly lower than in TW. As the salt concentration in drinking water increases, damage to testicular tissue. The MSW group demonstrating vacuolation of lining epithelial cells with pyknotic nuclei in the epididymis and necrosis and desquamation of spermatogenic cells in seminiferous tubules while HSW group displaying desquamated necrotic cells and giant cell formation in the epididymis, as well as damage to some of the seminiferous tubules and showed congestion, vacuolation of spermatogenic epithelium of seminiferous tubules, and desquamated necrotic spermatogenic epithelium. In conclusion, the salinity of the water has detrimental impacts on the sperm morphology, viability and concentration, hormones and antioxidant levels in Barki rams.

POTENSI KANDUNGAN ANTIOKSIDAN RUMPUT KEBAR (Biophytum Petersianum Klotzsch) TERHADAP APOPTOSIS DAN GAMBARAN HISTOPATOLOGIS TESTIS PADA MENCIT (MUS MUSCULUS) YANG DIPAPAR 2,3,7,8-Tetrachlorodibenzo-p-dioxin

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most toxic compound in the dioxin group. This compound is a pollutant for the environment and very harmful to human health and enter the body through the mucous membranes in the mouth and the respiratory tract and can be transmitted through the placenta and lactation. The aim of this study was to know the antioxidantpotency of to resolve reproduction disturbance caused by TCDD exposure.Thirty Balb/C male mice were divided into five different groups, the negative control group, a positive control group exposed to TCDD at a dose of 7 μg/kg BW, P1 group of groups exposed to TCDD doses of 7 μg / kg BW and given Biophytum petersianum extract 0.05mg/gBB/day, group P2 group exposed to TCDD doses of 7μg/kgBW and given 0.080mg/gBB/day, and group P3 were exposed to TCDD dose of 7μg/kgBB and given the extract of Biophytum petersianum 0.135mg/gBB/day during day 2 to day 55. On the 56th day the mice were sacrificed and apoptotic examination and spermatogenic cell histopathological features were performed on the testis. The results showed that: P2 (0,433 ± 0,497; p<0,05) and P3 (0,200 ± 0,000 p<0,05) groups were the most effective group in decreasing spermatogenic cell apoptosis compared to positive control group (2,933 ± 1,5832 p<0,05). The Johnsen score result showed that P2 (9,400 ± 0,420) and P3 (9,800 ± 0,253) groups improving the histopathologic picture of spermatogenic cells in seminiferous tubules compared to positive control group (7,20 ± 0,400) p<0,05. Conclusion of this study were Biophytum petersianum is effective to solve reproduction disturbances caused by exposure of TCDD and the P3 group is the most effective group.

Social Jetlag and Damage to Male Reproductive System: Epidemiological Observation in European and Chinese Populations and Biochemical Analyses in Mice

Social jetlag (SJ), discrepancy from inner clock for work and non-work-related reasons, is prevalent worldwide and has been associated with decreased fertility in women, but not in men. In the current study, we firstly extracted the national rate of rotating shift work (RSW) in men of reproductive age (15-49 years) from 35 European countries from the European Working Conditions Survey. Regression analysis revealed an inverse correlation between RSW and the national fertility rate (as reported by the World Bank; P < 0.001). For every 10% increase in RSW rate, fertility rate decreased by 0.114 (equivalent to 11.4 fewer births per 100 women). Then we compared 16 semen/reproductive-hormone parameters of RSW workers against day workers in 1346 reproductive-age Chinese men. RSW was associated with 1.26 (95%CI 1.05-1.52) fold risk of low sperm count. The SJ-sperm correlation was also observed in a cohort of 796 unemployed reproductive-age Chinese undergraduates: each hour non-work-related SJ between school days and days off was associated with 1.16 (95%CI 1.02-1.31) fold risk, and sperm count increased during follow up in men who reduced SJ after baseline. At last, a mouse model subjected to photoperiod shifting was established to simulate SJ and the related sperm decrease. Apoptosis analysis suggested the damage originated in spermatogenic cells in seminiferous tubules. Differentially expressed gene analysis showed that photoperiod shifting down-regulated marker genes of post-spermatocyte stage cells. The most enriched functional pathway was homologous recombination, which happened in spermatocytes during meiosis. Our study suggests that SJ is associated with male reproductive damage.

Leaf and Petiole Extract of Centella Asiatica are Potential for Antifertility and Antimicrobial Material

Ethanol extract of leaves and petiole of pegagan (Centella asiatica) is potential as antimicrobial and antifertility. This study was to investigate whether ethanol extracts of leaf and petiole of Centella asiatica has potential as an antimicrobial and can decrease sperm quality and spermatogenic cells of mice. This was a randomized controlled study using Centella asiatica plant from which we made ethanol extract. The extract of the leaves and petiole of Centella asiatica was subjected to antimicrobial test using the bacteria S. aureus, S. epidermidis, and A. aerogenes. Then the observation was done after a 24-hour-old bacterial cultures by measuring the diameter of the clear zone in mm. Thereafter, the extract was given to randomly grouped mice that were divided into two groups receiving the leaf extract in one group and the petiole exract in another for 35 days. On day 36 the mice were sacrificed for spermatozoa quality and testicular histology examination. Statistical analyses used in this study were ANOVA and LSD tests. There was inhibition zone difference between leaf and petiole (p=0.000). Inhibition zone between bacteria S. epidermidis, S. aureus and A. aerogenes showed no difference (p=0.198). Inter-dose inhibition zone showed difference (p=0.000). Centella asiatica leaf extract showed inhibition zone better than that of Centella asiatica petiole extract. In spermatozoa quality test, there was effect of Centella asiatica leaf extract on sperm motility (p&lt;0.05), viability (p&lt;0.05), morphology (p&lt;0.05) and concentration (p&lt;0.05). No effect of pegagan petiole extract was found on sperm motility (p&lt;0.05), viability (p&lt;0.05), abnormal morphology (p&lt;0.05) and concentration (p&lt;0.05). Highest degradation of spermatozoa quality was obtained from Centella asiatica petiole extract at a concentration of 150 mg/kg bw. Spermatogenic cell test results showed no effect of Centella asiatica leaf extract in decreasing spermatogonia (p=0.000), spermatocytes (p=0.000), and spermatids (p=0.000) counts. No effect of pegagan petiole extract in decreasing the number of spermatogonia, spermatocytes and spermatids. The highest reduction spermatogenic cells of seminiferous tubules was obtained by Centella asiatica petiole extract in a concentration of 150 mg/kg bw. In conclusion: Centella asiatica leaf and petiole extract has antimicrobial potency as contra-ceptives

Immunohistochemical investigation of testicular morphogenesis in offspring rats, developing under the influence of maternal hypothyroidism.

Background. Thyroid disorders are currently among the most widespread endocrine pathologies, influencing morphogenesis and histophysiology of multiple organs. Objective . The aim of present investigation was to study the impact of maternal hypothyroidism on the testicular development of rats progeny using immunohistochemical detection of cell proliferation marker Ki-67 and vascular endothelium growth factor VEGF. Methods. Hypothyroidism was induced by daily food supplementation of experimental group rats with 10 mg/kg of anti-thyroid drug Merkazolil during two weeks before pregnancy, throughout the whole gestation and lactation periods. Testes of their progeny on postnatal days 1st, 10th, 20th and 40th were removed after euthanasia, fixed in Bouin’s fluid, embedded in paraffin and subjected to immunohistochemical investigation. Results. On postnatal day 1st in control group rats Ki-67 labeling was restricted to cytoplasmic granularity of gonocytes – fetal germ cells, located centrally in within the seminiferous cords. Under hypothyroid conditions additionally to gonocytes Ki-67 binding was detected in macrophages and Leydig cells, number of both significantly elevated. On postnatal day 10th control group animals demonstrated moderate labeling of Sertoli cells, spermatogonia and gonocytes being completely non-reactive. In hypothyroidism affected testes was revealed disperse distribution of Ki-67 marker with predominant accumulation on the periphery of seminiferous cords and in Leydig cells. On postnatal day 20th Ki-67 reactivity was detected in spermatogenic cells of seminiferous tubules. In hypothyroidism affected animals cytoplasmic granularity labeling of maturating spermatogenic cells was higher compared to control, though seminiferous tubules were smaller in diameter and lacking luminae. On postnatal day 40th Ki-67 labeled predominantly spermatogonia, immunoreactivity of which enhanced under hypothyroid conditions. Distribution of VEGF label was generally similar to that of Ki-67, indicating a direct correlation of cell proliferation intensity on saturation of tissues with oxygen. Unlike Ki-67, VEGF exposed strong reactivity with acrosomal granules of early spermatids on postnatal day 20th, as well as more intense spermatogenic cells labeling compared to Ki-67. Conclusion. Testicular morphogenesis in offspring rats, developing under maternal hypothyroidism, was accompanied by the increased immunoreactivity of spermatogenic cell cytoplasm on postnatal days 20th and 40th with Ki-67 and VEGF, as well as acrosomal granules labeling with VEGF. This trend was supplemented with the increased count of macrophages and Leydig cells in testicular interstitium, decreased count and size of spermatogenic cells, damage of their syncytial complexes, all these signs encompassing degenerative changes in seminiferous tubules.

Bi-allelic Recessive Loss-of-Function Variants in FANCM Cause Non-obstructive Azoospermia

Infertility affects around 7% of men worldwide. Idiopathic non-obstructive azoospermia (NOA) is defined as the absence of spermatozoa in the ejaculate due to failed spermatogenesis. There is a high probability that NOA is caused by rare genetic defects. In this study, whole-exome sequencing (WES) was applied to two Estonian brothers diagnosed with NOA and Sertoli cell-only syndrome (SCOS). Compound heterozygous loss-of-function (LoF) variants in FANCM (Fanconi anemia complementation group M) were detected as the most likely cause for their condition. A rare maternally inherited frameshift variant p.Gln498Thrfs∗7 (rs761250416) and a previously undescribed splicing variant (c.4387-10A>G) derived from the father introduce a premature STOP codon leading to a truncated protein. FANCM exhibits enhanced testicular expression. In control subjects, immunohistochemical staining localized FANCM to the Sertoli and spermatogenic cells of seminiferous tubules with increasing intensity through germ cell development. This is consistent with its role in maintaining genomic stability in meiosis and mitosis. In the individual with SCOS carrying bi-allelic FANCM LoF variants, none or only faint expression was detected in the Sertoli cells. As further evidence, we detected two additional NOA-affected case subjects with independent FANCM homozygous nonsense variants, one from Estonia (p.Gln1701∗; rs147021911) and another from Portugal (p.Arg1931∗; rs144567652). The study convincingly demonstrates that bi-allelic recessive LoF variants in FANCM cause azoospermia. FANCM pathogenic variants have also been linked with doubled risk of familial breast and ovarian cancer, providing an example mechanism for the association between infertility and cancer risk, supported by published data on Fancm mutant mouse models.

Effect of Spirulina Platensis on The Number of Spermatogenic Cells in The Seminiferous Tubules of Rat (Rattus Norvegicus) with Excessive Physical Exercise

The aim of this study was to investigate the effect of Spirulina platensis extract on the number of spermatogenic cells of rats with excessive physical exercise. Excessive physical exercise was achieved through one hour of swimming for 35 days. Twenty male rats were divided into five groups i.e (1) C-, control group, (2) C+, a group was only given swimming, (3) T1, a group was given 300 mg/kg BW of Spirulina platensis and swimming, (4) T2, a group was given 600 mg/kg BW of Spirulina platensis and swimming, and (5) T3, a group was given 1200 mg/kg BW of Spirulina platensis and swimming. Spirulina platensis extract was given orally once a day before swimming. ANOVA test followed with Duncan test showed that the number of spermatogenic cells significantly different among treatments (p&lt;0.05). Excessive physical exercise was able to influence on the male reproduction system by declined on the number of spermatogenic cells in seminiferous tubules of male rat. The conclusion of this study was dose of 1200 mg/kg BW of Spirulina platensis extract could maintain the number of spermatogenic cells of male rat after excessive physical exercise. Keywords : Rattus norvegicus, Spirulina platensis, excesive physical exercise, spermatogenic cells

Effect of low-dose zearalenone exposure on reproductive capacity of male mice

Zearalenone (ZEA), a kind of nonsteroidal mycotoxin with estrogenic effects, can influence animal reproductive capacity through interfering with estrogen signaling pathway. Previous studies have shown exposure to ZEA at high doses (higher than No-Observed Effect Level, NOEL) had a significant impact on mouse sperm quality and pregnant rate, but little is known about the effect of exposure to ZEA at low doses (lower than NOEL) on mouse reproductive capacity. This study evaluated the effects of exposure to low-dose ZEA on mouse spermatogenesis and semen quality. Male mice (CD-1) of 21days were exposed to ZEA at 20, or 40μg/kg body weight for 14, 28 or 42days. After exposure to ZEA for 14days, the spermatogenic cells in seminiferous tubules were declined dose-independently; however in groups treated by ZEA for 28days, the spermatogenic cells were declined dose-dependently. Moreover, after treatment for 28days or 42days, the DNA double stand break (DSB) in spermatogenic cells were increased in a dose-dependent manner in treated groups. Compared with the control group, the sperm concentration, viability, motility, and hyperactive rate in treated groups were decreased dose-dependently and time-dependently. Meanwhile, deformity and mortality rate of sperm in treated groups were increased remarkably dose-dependently too. In conclusion, low dose ZEA impaired male reproductive capacity especially in spermatogenesis and semen quality of mouse.

468 The application of busulfan to inhibit the spermatogenesis in chicken testis

Spermatogonia cells are considered as a valuable genetic material to produce individuals with desired traits in the framework of preservation of animal and poultry gene pool (cryobanks) and genetic modification of their genome (transgenesis). This technology is based on the introduction of donor spermatogonia into the testes of male recipients and the following obtaining the exogenous sperm to produce progeny with desired characteristics. A key step of this technology is the removal of own spermatogenic cells (spermatogenesis inhibition) in the male recipient. We have optimized the method to inhibit the spermatogenesis in chicken testes using alkylating agent busulfan. The multiple injections of busulfan at the concentration from 40 to 100 mg per 1 kg of body weight was performed directly into the parenchyma of testes of six month old chicken males (n = 16). In order to detect the presence of spermatogenic cells in seminiferous tubules, we carried out the histological studies of testes in one month after injection. We observed the decrease in diameters and the number of spermatogenic cells at the cross sectional area of the seminiferous tubules in the experimental group as compared to the control. When using busulfan at concentration of 40 mg/kg the number of cells averaged 35±3 cells per seminiferous tubule, which was 32.8 times less as compared to the control (1149±95). At higher concentrations of busulfan the number of cells decreased and averaged 20±2, 12±0.5 and 9±0.4 cells at concentration of 60, 80 and 100 mg/kg, repectively. Analysis of cell types of the seminiferous tubules revealed that mainly the Sertoli cells and spermatogonia represented the cell population. Moreover, with increasing concentrations of busulfan the ratio of spermatogonia was significantly reduced. The ratio of Sertoli and spermatogonia cells after introduction of busulfan into the testes at a concentration of 40, 60, 80 and 100 mg/kg was 57 and 43%, 75 and 25%, 95 and 5 % and 96 and 4%, respectively. However, the application of busulfan at a concentration 100 mg/kg led to high mortality of chicken. Thus, we demonstrated that the optimal concentration of busulfan to kill the spermatogonial cells was 80 mg/kg. The complete inhibition of spermatogenesis was observed in 9-10 months after introduction of busulfan at this concentration. The study was supported by the Russian Science Foundation within Project no.16–16–10059.

Investigation of the effects of kisspeptin-10 in methionine-induced lipid peroxidation in testicle tissue of young rats.

Disruption of the balance oxidants, antioxidants cause various pathophysiological conditions such as lipid peroxidation, protein degradation, or DNA damage. We have examined possible effects of kisspeptin-10 on the structural damage produced by methionine-induced lipid peroxidation in testicle tissue of young rats. Kisspeptin-10 did not significantly affect spermatogenic cells in seminiferous tubules. Testosterone levels decreased in the methionine group as compared with the control group but without statistical significance. Luteinizing hormone levels decreased in the methionine group as compared with the control group (P<0.001). Catalase enzyme activity increased in the kisspeptin-10 group (P<0.01) as compared with the other groups. Catalase mRNA expression was decreased in the methionine group as compared with the kisspeptin group (P<0.001). Total superoxide dismutase enzyme activity and superoxide dismutase mRNA expression were increased in the kisspeptin group as compared with the methionine group (P<0.05). In conclusion, kisspeptin treatment may protect the structure of spermatogenic cells against methionine-induced damage.

Radioprotective potential of melatonin against ⁶⁰Co γ-ray-induced testicular injury in male C57BL/6 mice.

BackgroundMelatonin, the chief secretary product of pineal gland, is a strong free radical scavenger and antioxidant molecule. The radioprotective efficacy and underlying mechanisms refer to its antioxidant role in somatic cells. The purpose of this study, therefore, was to investigate the prophylactic implications of melatonin against γ-ray-induced injury in germinal cells (testes). C57BL/6 male mice were administered melatonin (100 mg/kg) intra-peritoneally 30 min prior to a single dose of whole-body γ-irradiation (5 Gy, 1 Gy/minute) using 60Co teletherapy unit. Animals were sacrificed at 2h, 4h and 8h post-irradiation and their testes along with its spermatozoa taken out and used for total antioxidant capacity (TAC), lipid peroxidation, comet assay, western blotting and sperm motility and viability. In another set of experiment, animals were similarly treated were sacrificed on 1st, 3rd, 7th, 15th and 30th day post-irradiation and evaluated for sperm abnormalities and histopathological analysis.ResultsWhole-body γ-radiation exposure (5 Gy) drastically depleted the populations of spermatogenic cells in seminiferous tubules on day three, which were significantly protected by melatonin. In addition, radiation-induced sperm abnormalities, motility and viability in cauda-epididymes were significantly reduced by melatonin. Melatonin pre-treatment significantly inhibited radiation-induced DNA strands breaks and lipid peroxidation. At this time, radiation-induces activation of ATM-dependent p53 apoptotic proteins-ATM, p53, p21, Bax, cytochrome C, active caspase-3 and caspases-9 expression, which were significantly reversed in melatonin pre-treated mice. This reduced apoptotic proteins by melatonin pre-treatment was associated with the increase of anti-apoptotic-Bcl-x and DNA repair-PCNA proteins in irradiated mice. Further, radiation-induced decline in the TAC was significantly reversed in melatonin pre-treated mice.ConclusionsThe present results indicated that melatonin as prophylactic agent protected male reproductive system against radiation-induced injury in mice. The detailed study will benefit in understanding the role of melatonin in modulation of radiation-induced ATM-dependent p53-mediated pro-vs.-anti apoptotic proteins in testicular injury. These results can be further exploited for use of melatonin for protection of male reproductive system in radiotherapy applications involving hemibody abdominal exposures.Electronic supplementary materialThe online version of this article (doi:10.1186/s12929-015-0156-9) contains supplementary material, which is available to authorized users.

Varicocele-Caused Progressive Damage in Bilateral Testis and Sertoli Cell-Only Syndrome in Homolateral Testis in Rats

BackgroundWe aimed to investigate whether varicocele (VC) in rats can cause Sertoli cell-only syndrome (SCOS).Material/MethodsForty adolescent SD rats were randomly divided into 4 groups: 4-weeks control group, 4-weeks experimental group, 12-weeks control group, and 12-weeks experimental group. Left varicocele models were introduced by partially ligating left kidney veins for the experimental groups, and the sham surgery groups as controls were executed with exactly the same surgery as in the experimental groups except for the ligation. Rats in control and experimental groups for 4 and 12 weeks were killed after laparotomy at 4 and 12 weeks, respectively, the testes were taken out and fixed in fixative containing 4% polyformaldehyde, then were stained by hematoxylin and eosin (HE). The density and viability of sperm were analyzed by computer-aided sperm analysis.ResultsCompared with rats in 4-weeks and 12-weeks control group, histological structures of bilateral testes in both experimental groups were impaired, most of them showing as focal focuses. The pathological changes of testes in rats of the 12-weeks experimental group were bilateral, and included atrophy of seminiferous tubules, turbulence of spermatogenic cells in seminiferous tubules, defluvium of most spermatogenic cells, abortion of spermatogenesis, and degradation of spermatogenic epithelia. One rat in the 12-weeks experimental group was shown having SCOS, with the spermatogenic cells in seminiferous tubules completely flaked, degraded, or absent, and only Sertoli cells lined the seminiferous tubules.ConclusionsLaboratory VC caused progressive impairment of homolateral testes, and SCOS could be induced when the damage was severe. Our results indicate that asthenozoospermia, azoospermia, and SCOS can be prevented by the earlier treatment of VC.

Characterization of a Novel Human Testis-Specific Gene: Testis Developmental Related Gene 1 (TDRG1)

Spermatogenesis is a highly coordinated physiological process that requires the correct expression and functions of thousands of developmentally regulated genes. The regulation of spermatogenesis is not well defined, since majority of the related genes have neither been identified nor fully characterized. Hence, it is meaningful to identify and characterize these genes to reveal the mechanism underlying spermatogenesis. In this study, using digital differential display, we identified a novel human testis-specific gene, testis developmental related gene 1 (TDRG1, GenBank DQ168992), via electronic subtraction of human testis UniGene databases from those of non-reproductive tissues. The transcript of the TDRG1 gene has an open-reading frame that encodes 100 amino acids. We next prepared the anti-TDRG1 monoclonal antibody 10B6 and confirmed that it specifically recognizes an 11-kDa protein in the tissue extracts from an adult human testicular sample (age 31 years) by Western blot analysis. RT-PCR coupled with immunohistochemistry of human tissues demonstrated that TDRG1 is exclusively expressed in the testis but not in any other non-reproductive tissues. TDRG1 is mainly located in spermatogenic cells in seminiferous tubules of adult testis. Furthermore, TDRG1 shows the highest expression level in human post-puberty testis, with the expression levels decreasing afterwards with aging. Importantly, TDRG1 mRNA is undetectable in the fetal testis, as judged by RT-PCR. In conclusion, TDRG1 is a developmentally regulated testicular-specific gene. We suggest that TDRG1, a newly identified testis-specific gene, may play important roles in human spermatogenesis.

Effects on rat testes of the thiosemicarbazone derivative Schiff base (4-(1-phenylmethylcyclobutane-3-yl)-2-(2-hydroxybenzylidenehydrazino)thiazole) and its cadmium(II) complex

The aim of this study was to investigate structural and biochemical changes in testes of rats treated with the thiosemicarbazone derivative thiazole ring Schiff base, (4-(1-phenyl-methylcyclobutane-3-yl)-2-(2-hydroxybenzylidene-hydrazino) thiazole (L), and its Cd(II) complex (CdL(2)). The animals were divided into three groups. Group I was designated as control. The rats in groups II and III were injected subcutaneously with L or CdL(2) respectively at 150-mg kg(-1) doses at 3-day intervals for 15 days. At the end of the study, blood samples were collected for biochemical analysis, and testes were removed for histological examinations. Serum levels of vitamin A, E and MDA of the L-injected group were similar to the control group. While CdL(2) treatment decreased serum vitamin A and E levels, it increased the MDA level compared to other groups. Histologically, the testes structures of L-treated animals were similar to the control. Spermatogenic cells in seminiferous tubules of CdL(2)-treated animals displayed necrosis. Nuclei of spermatogonia and primary spermatocytes were pyknotic and heterochromatic. Homogenous pink particles were present in place of the spermatids. The interstitial areas were oedematous and intertubular vessels were plugged. In conclusion, the present results indicate that L does not cause biochemical and morphological alterations, but its Cd(II) complex has degenerative effects in normal rat testes.

Expression and functional characterization of the adhesion molecule spermatogenic immunoglobulin superfamily in the mouse testis.

Spermatogenic immunoglobulin superfamily (SgIGSF) is a mouse protein belonging to the immunoglobulin superfamily expressed in the spermatogenic cells of seminiferous tubules. We produced a specific polyclonal antibody against SgIGSF. Western blot analysis of the testes from postnatal developing mice using this antibody demonstrated multiple immunopositive bands of 80-130 kDa, which increased in number and size with the postnatal age. Enzymatic N-glycolysis caused reduction in the size of these bands to 70 kDa, indicating that SgIGSF is a glycoprotein and its glycosylation pattern and extent are developmentally regulated. Immunohistochemical analysis of the adult testis demonstrated that SgIGSF was present in the spermatogenic cells in the earlier steps of spermatogenesis and increased in amount from intermediate spermatogonia through zygotene spermatocytes but was diminished in the steps from early pachytene spermatocytes through round spermatids. After meiosis, SgIGSF reappeared in step 7 spermatids and was present in the elongating spermatids until spermiation. The immunoreactivity was localized primarily on the cell membrane. Consistent with the findings in adult testes, the analysis of the developing testes revealed that SgIGSF was expressed separately in the spermatogenic cells in earlier and later phases. Sertoli cells had no expression of SgIGSF, whereas both SgIGSF immunoprecipitated from the testis lysate and produced in COS-7 cells was shown to bind to the surface of Sertoli cells in primary culture. These results suggested that SgIGSF on the surface of spermatogenic cells binds to some membrane molecules on Sertoli cells in a heterophilic manner and thereby may play diverse roles in the spermatogenesis.

Localization and physiological implication of aldose reductase and sorbitol dehydrogenase in reproductive tracts and spermatozoa of male rats.

The polyol metabolizing pathway, which consists of two enzymes, aldose reductase (AR) and sorbitol dehydrogenase (SDH), converts glucose to fructose. The enzymatic activities, expression, and localization of AR and SDH were studied in reproductive tracts and spermatozoa of male rats by immunohistochemistry, Western blotting, and enzyme assays. Immunoreactivity to an AR antibody was observed mainly in epithelia of epididymis, seminal vesicle, vas deferens, and prostate gland in adult rats. Similar staining profiles were observed for these tissues when an SDH antibody was used. However, in testis, the cells that express these 2 enzymes differed; whereas AR was expressed in Sertoli cells and to lesser extent in spermatogenic cells, SDH was detected in spermatogenic cells of seminiferous tubules. This cell type-specific gene expression was confirmed in primary cultured cells isolated from rat testes. SDH protein levels were higher during spermatid elongation, and large amounts of SDH were carried over to the spermatozoa. Because one of the functions of members of the aldo-keto reductase superfamily is to detoxify harmful carbonyl compounds, an intrinsic function of AR in Sertoli cells may be to catalyze the reduction of cytotoxic metabolites, such as lipid peroxidation products and steroid hormones, which are produced during spermatogenesis. Because uterine fluid and seminal plasma both contain sorbitol, it is likely that SDH in spermatozoa converts sorbitol to fructose for use as an energy source.

Immunohistochemical Localization of Follistatin in Rat Tissues.

We have used immunohistochemistry to localize follistatin/activin-binding protein in adult male and female rats. A polyclonal antibody directed against a follistatin peptide (residues 123-134) was used as a specific immunologic probe. Intense and specific follistatin immunoreactivity was evident in spermatogenic cells of seminiferous tubules in the testis. The predominant staining was in nuclei of spermatocytes and spermatids, but no immune reaction was observed in spermatogonia or spermatozoa. Moderate immunoreactivity was detected in Leydig cells. Sertoli cells were follistatin-negative. Significant immunoreactivity was evident in ovarian granulosa cells. The intensity of the staining changed with follicle development: no immunoreactivity was observed in granulosa cells of primordial to primary follicles, but the cells of secondary to Graafian follicles displayed moderate to strong staining and finally luteal cells of the corpus luteum became negative. The epithelial lining of the oviduct and the smooth muscle of the myometrium of the uterus were intensely immunoreactive. Immunoreactive follistatin staining was present in the pituitary: a group of round-shaped cells were specifically stained. Immunostainable follistatin was visible in the epithelial layers of renal tubules with moderate to strong staining reactivity. Hepatic cells in the liver demonstrated homogeneous immunoreactivity from moderate to strong. The cortex of the adrenal gland, white pulp of the spleen and the brain cortex were also stained weakly but distinctly with the antiserum. In conclusion, immunoreactive follistatin is widespread in rat tissues, suggesting that follistatin/activin-binding protein is a ubiquitous protein, regulating a wide variety of activin actions.