Rat Germinal Cells Research Articles

In Vitro Effect of Gold or Silver Nanoparticles on Meiotic and Postmeiotic Fractions of Rat Germinal Cells

The cytotoxicity of Au or Ag nanoparticles (NPs) on rat germinal cells was investigated in vitro. Rat germ cells separated by the STAPUT method in two different populations, pachytene spermatocytes (F5) and round spermatids (F3), were incubated (37°C, 60'-120') with 60 μM, 125 μM, 250 μM and 500 μM of Au/Ag-NPs. Cell viability was assessed with the Eosin Y test. Au or Ag-NPs were investigated with FEG-STEM/EDS and TEM. A dose-dependent effect on the viability of F3 and F5 populations was observed after incubation with Au-NPs or Ag-NPs (P<0.001). A significant de- crease in cell viability was observed in F5 fractions compared to F3 fractions (P<0.05), except for the samples treated with 60μM of Au-NPs, and the decrease was also significant in all the samples treated with Ag-NPs compared to those incu- bated with Au-NPs (P< 0.05). Au-NPs were localized in spermatocytes and spermatids whereas Ag-NPs were undetect- able. In conclusion, Au- NPs and Ag-NPs seem to exert a negative effect on rat germ cells, particularly on spermatocytes, that appeared significantly more compromised than spermatids. Further research is needed, mainly to carefully explore the possible genotoxicity of these NPs on germinal cells.

ANTIOXIDANT EFFECT OF RESVERATROL IN HUMAN SPERMATOZOA AND IN RAT GERMINAL CELLS

Objective: To assess the antioxidant activity of Resveratrol (3,5,4&rsquo;-trihydroxystilbene, RES) after induction of lipid peroxidation LPO) in human spermatozoa and in immature rat germinal cells. Materials and Methods: Ejaculated human spermatozoa, selected by swim up, have been incubated with tert-Butylhydroperoxide and tert-Butylhydroperoxide-RES. The localization of LPO has been performed using the probe C11-BODIPY581/591. The same assays were carried out on pachytene spermatocytes and round spermatids obtained from three Wistar rats 35 days of age. The two cellular fractions were achieved after enzymatic digestion with collagenase and subsequent fractionation on bovine serum albumin 0.5-3% gradient (STAPUT). The ultrastructure of all samples was assessed by transmission electron microscopy (TEM). Results: The midpiece of sperm tail and the whole plasma membrane of germ cells were the target of LPO. TEM analysis of sperm, quantitatively elaborated by a mathematical formula, showed a significantly lower percentage of necrosis in the samples treated with RES (P&lt;0.01); as regards rat germinal cells, necrosis features (cytoplasmic vacuoles, disrupted chromatin and broken plasma membrane) were mainly evident in the meiotic fraction without RES. Conclusions: RES, found in the skins of grape, reduces the damage induced by oxidative stress in human sperm and rat testicular germ cells; in particular spermatids appeared to be less sensitive to oxidative damage compared with spermatocytes.

Effect of trans-resveratrol on induced oxidative stress in human sperm and in rat germinal cells

Resveratrol is a phytoalexin with antioxidant properties. We evaluated resveratrol toxicity in swim-up selected human sperm and in rat spermatocytes and spermatids separated by the STAPUT method. Resveratrol antioxidant activity was tested against lipid peroxidation (LPO) induced by tert-butyl hydroperoxide. LPO was evaluated using the C11-BODIPY 581/591 probe and transmission electron microscopy in samples incubated with and without resveratrol. LD50 for human sperm and rat spermatids was 50 μM; spermatocytes were more sensitive to resveratrol cytotoxicity. Sperm motility increased progressively at 30 μM, 15 μM and 6 μM. 15 μM resveratrol acts against LPO, preserving sperm chromatin and plasma membranes. LPO were more marked in spermatocytes than in spermatids and the effect of resveratrol was more evident in spermatocytes. In this study, the scavenger properties of resveratrol were demonstrated in vitro in human sperm and rat germ cells, thus resveratrol could be added to the media used in assisted reproduction techniques and cryopreservation when oxidative stress is exacerbated.

ANTIOXIDANT EFFECT OF RESVERATROL IN HUMAN SPERMATOZOA AND IN RAT GERMINAL CELLS

Objective: To assess the antioxidant activity of Resveratrol (3,5,4’-trihydroxystilbene, RES) after induction of lipid peroxidation LPO) in human spermatozoa and in immature rat germinal cells. Materials and Methods: Ejaculated human spermatozoa, selected by swim up, have been incubated with tert-Butylhydroperoxide and tert-Butylhydroperoxide-RES. The localization of LPO has been performed using the probe C11-BODIPY581/591. The same assays were carried out on pachytene spermatocytes and round spermatids obtained from three Wistar rats 35 days of age. The two cellular fractions were achieved after enzymatic digestion with collagenase and subsequent fractionation on bovine serum albumin 0.5-3% gradient (STAPUT). The ultrastructure of all samples was assessed by transmission electron microscopy (TEM). Results: The midpiece of sperm tail and the whole plasma membrane of germ cells were the target of LPO. TEM analysis of sperm, quantitatively elaborated by a mathematical formula, showed a significantly lower percentage of necrosis in the samples treated with RES (P<0.01); as regards rat germinal cells, necrosis features (cytoplasmic vacuoles, disrupted chromatin and broken plasma membrane) were mainly evident in the meiotic fraction without RES. Conclusions: RES, found in the skins of grape, reduces the damage induced by oxidative stress in human sperm and rat testicular germ cells; in particular spermatids appeared to be less sensitive to oxidative damage compared with spermatocytes.

ANTIOXIDANT EFFECT OF RESVERATROL IN HUMAN SPERMATOZOA AND IN RAT GERMINAL CELLS

Objective: To assess the antioxidant activity of Resveratrol (3,5,4&rsquo;-trihydroxystilbene, RES) after induction of lipid peroxidation LPO) in human spermatozoa and in immature rat germinal cells. Materials and Methods: Ejaculated human spermatozoa, selected by swim up, have been incubated with tert-Butylhydroperoxide and tert-Butylhydroperoxide-RES. The localization of LPO has been performed using the probe C11-BODIPY581/591. The same assays were carried out on pachytene spermatocytes and round spermatids obtained from three Wistar rats 35 days of age. The two cellular fractions were achieved after enzymatic digestion with collagenase and subsequent fractionation on bovine serum albumin 0.5-3% gradient (STAPUT). The ultrastructure of all samples was assessed by transmission electron microscopy (TEM). Results: The midpiece of sperm tail and the whole plasma membrane of germ cells were the target of LPO. TEM analysis of sperm, quantitatively elaborated by a mathematical formula, showed a significantly lower percentage of necrosis in the samples treated with RES (P&lt;0.01); as regards rat germinal cells, necrosis features (cytoplasmic vacuoles, disrupted chromatin and broken plasma membrane) were mainly evident in the meiotic fraction without RES. Conclusions: RES, found in the skins of grape, reduces the damage induced by oxidative stress in human sperm and rat testicular germ cells; in particular spermatids appeared to be less sensitive to oxidative damage compared with spermatocytes.

Lead-, Cadmium-, and Arsenic-Induced DNA Damage in Rat Germinal Cells

Toxic agents can interfere with the male reproductive system at many targets. One of the major unresolved questions concerning male infertility is identification of its molecular origins. Clinical and animal studies indicate that abnormalities of spermatogenesis result from exposure to three toxic metals (lead acetate, cadmium chloride, and arsenic trioxide), but the effects on primary spermatocyte DNA of the male rat after chronic exposure to these metals have not been identified. The aims of this study were to analyze, in three independent experiments, the DNA damage induced by lead (Pb), cadmium (Cd), and arsenic (As) in rat germinal cells during three time periods, and to determine the relationship between DNA damage and blood Pb, blood Cd, and urine As levels. For lead acetate and cadmium chloride experiments, blood was collected by cardiac puncture, while for arsenic trioxide a 24-h urine sample was collected. Afterward, the animals were sacrificed by decapitation. Pachytene spermatocytes from rat testes were purified by trypsin digestion followed by centrifugal elutriation. After establishment of cell purity and viability, DNA damage (tail length) was measured employing a single cell gel/comet assay. Significant DNA damage was found in primary spermatocytes from rats with chronic exposure (13 weeks) to toxic metals. In conclusion, these findings indicate that exposure to toxic metals affects primary spermatocyte DNA and are suggestive of possible direct testicular toxicity.

Dual role for poly(ADP-ribose)polymerase-1 and -2 and poly(ADP-ribose)glycohydrolase as DNA-repair and pro-apoptotic factors in rat germinal cells exposed to nitric oxide donors

We describe the involvement of poly(ADP-ribose)polymerase 1 and 2 (PARP-1 and -2) and poly(ADP-ribose)glycohydrolase (PARG) in the response of rat germinal cells to the action of the NO donors, 3-morpholino-sydnonimine (SIN-1) and spermine nonoate (SNO). Primary spermatocytes and round spermatids showed a differential sensitivity to DNA damage induced by acute exposure to SIN-1 and SNO. Spermatocytes were able to repair DNA damage caused by the release of NO from SNO but neither spermatocytes nor spermatids could recover from the release of NO and O 2 − from SIN-1. Addition of the PARPs inhibitor, 3-aminobenzamide, and the PARG inhibitor, gallotannin (GT), to germ cell cultures impaired DNA repair significantly. Consistent with the DNA repair seen in primary spermatocytes, both SIN-1 and SNO induced PARPs activation in these cells. In the case of SIN-1, there was an immediate but transient response while SNO induced a delayed but more sustained increase in PARPs activity. Chronic exposure of spermatocytes to SIN-1 and SNO, however, committed the cells to apoptosis, which coincided with proteolysis of PARP-1. The data indicate a dual role for PARPs and PARG in germinal cells as key proteins in processes that sense and repair DNA damage as well as in the commitment to apoptosis following prolonged oxidative stress.

Dual role for poly(ADP-ribose)polymerase-1 and -2 and poly(ADP-ribose)glycohydrolase as DNA-repair and pro-apoptotic factors in rat germinal cells exposed to nitric oxide donors

We describe the involvement of poly(ADP-ribose)polymerase 1 and 2 (PARP-1 and -2) and poly(ADP-ribose)glycohydrolase (PARG) in the response of rat germinal cells to the action of the NO donors, 3-morpholino-sydnonimine (SIN-1) and spermine nonoate (SNO). Primary spermatocytes and round spermatids showed a differential sensitivity to DNA damage induced by acute exposure to SIN-1 and SNO. Spermatocytes were able to repair DNA damage caused by the release of NO from SNO but neither spermatocytes nor spermatids could recover from the release of NO and O 2 − from SIN-1. Addition of the PARPs inhibitor, 3-aminobenzamide, and the PARG inhibitor, gallotannin (GT), to germ cell cultures impaired DNA repair significantly. Consistent with the DNA repair seen in primary spermatocytes, both SIN-1 and SNO induced PARPs activation in these cells. In the case of SIN-1, there was an immediate but transient response while SNO induced a delayed but more sustained increase in PARPs activity. Chronic exposure of spermatocytes to SIN-1 and SNO, however, committed the cells to apoptosis, which coincided with proteolysis of PARP-1. The data indicate a dual role for PARPs and PARG in germinal cells as key proteins in processes that sense and repair DNA damage as well as in the commitment to apoptosis following prolonged oxidative stress.

Poly(ADPR) polymerase-1 and poly(ADPR) glycohydrolase level and distribution in differentiating rat germinal cells.

Poly(ADP-ribose)polymerase (PARP-1) and poly(ADP-ribose)glycohydrolase (PARG) are responsible for the transient poly(ADP-ribosyl)ation of proteins in eukaryotic cells. This biochemical reaction plays an active role in DNA replication and repair, transcription, cell differentiation and death. The aim of this study was to investigate the levels and the sub-cellular distribution of such enzymes in rat germinal cells at different stages of differentiation, i.e. in primary spermatocytes and round spermatids, representing meiotic and post-meiotic cells, respectively. The determination of the level of PARP-1 mRNA and protein revealed its higher expression in primary spermatocytes, thus implying that PARP-1 is one of the meiotic genes whose expression is requested at the pachytene phase of the meiosis. We also demonstrated that rat germinal cells contain both the forms of PARG (i.e. of 110 and 60 kDa) so far described in somatic cells. In our experimental system, the large PARG was present and active mainly in the nuclear fraction of primary spermatocytes, whereas round spermatids showed a higher level of the 60 kDa PARG in the post-nuclear fraction. Collectively, our data show a different expression level of PARP-1 and a different endocellular distribution of PARG and suggest a role for the poly(ADP-ribose) turnover in distinct pathways in meiotic and post-meiotic germinal cells.

Rat germinal cells require PARP for repair of DNA damage induced by γ-irradiation and H 2O 2 treatment

The ability of rat germinal cells to recover from genotoxic stress has been investigated using isolated populations of primary spermatocytes and round spermatids. Using a comet assay at pH 10.0 to assess single strand breakage (SSB) in DNA, it was found that a high level of damage was induced by 5 Gy gamma-irradiation and acute exposure to 50 microM H2O2. This damage was effectively repaired during a subsequent recovery period of 1-3 hours culture in vitro but repair was significantly delayed in the presence of the poly(ADP-ribose)polymerase (PARP) inhibitor 3-aminobenzamide (3-ABA). Immunofluorescence detection of PARP with specific antibodies localised the protein to discrete foci within the nucleus of both spermatocytes and spermatids. Poly(ADP-ribose) (pADPR) could also be detected in spermatid nuclei following gamma-irradiation or H2O2 treatment. Moreover, PARP activation occurs both in spermatocytes and spermatids left to recover after both genotoxic stresses. The NO donors, 3-morpholino-sydnonimine (SIN-1) and S-nitrosoglutathione (SNOG), caused significant SSBs in both spermatocytes and spermatids. The effects of SIN-1 could be prevented by exogenous catalase (CAT), but not superoxide dismutase (SOD), in the cell suspensions. SNOG-induced SSBs were insensitive to both CAT and SOD. It is concluded that DNA in spermatocytes and spermatids is sensitive to damage by gamma-irradiation and H2O2 and that efficient repair of SSBs requires PARP activity.

PARP response to genotoxic stresses in differentiating rat germinal cells

PARP response to genotoxic stresses in differentiating rat germinal cells

Metabolic Changes in the Poly(ADP-Ribosyl)ation Pathway of Differentiating Rat Germinal Cells

Endogenous levels of poly(ADP-ribose) and βNAD+ have been determined in rat male germinal cells at different stages of differentiation. The levels of both metabolites decreased progressively from primary spermatocytes to secondary spermatocytes and especially in spermatids. We have also determined the size and complexity of the ADP-ribose polymers synthesized in permeabilized germ cells. Polymers of different chain length and complexity were observed in cells incubated with different concentrations of [32P]βNAD+; short polymers characterized primary spermatocytes incubated with low βNAD+ concentration. In all cell fractions, polymers of over 20 residues in size were observed at high βNAD+ levels. Long polymers were associated with the sulfuric acid-insoluble proteins (nonhistone proteins such as PARP itself). By contrast, oligomers of 20 ADP-ribose units or less were found in the sulfuric acid-soluble proteins (histone proteins). We have also identified the main ADP-ribose protein acceptors formed in each cell type. In all cells examined, PARP appears to be extensively automodified. However, by far, the H1t variant of histone H1 appeared to be the preferred ADP-ribose target among the acid-soluble proteins separated by reverse-phase HPLC. Therefore, we conclude that an active protein–poly(ADP-ribosyl)ation system is concentrated in primary spermatocytes, based on a high level of PARP automodification accompanied by the preferential heteromodification of the histone H1 variant specifically expressed in the cells undergoing the pachytene phase of the meiotic division.

Expression of GnRH receptor in mouse and rat testicular germ cells.

The expression of gonadotrophin-releasing hormone receptor (GnRH-R) in germinal cells of mouse testis, whole testes, pituitary glands, and mouse ovaries was determined by means of Northern hybridization using a mouse GnRH-R [(32)P]-labelled cDNA probe. Also, the expression of GnRH-R in rat germinal cells, testis and pituitary gland was determined by Northern blot analysis using the same mouse-specific probe. Three receptor transcripts were detected in all cases. In mouse pituitary, ovary and testis, we found two GnRH-R transcripts in the proximity of 4.6-4.7 and 3.4 kb, as well as a 1.6 kb transcript in the pituitary and a 2.0-2.1 kb transcript in both the ovary and testis. Mouse germ cells also exhibited three GnRH-R transcripts of 4.7, 3.5 and 2.2 kb. Two distinct GnRH-R transcripts were also detected in the rat pituitary (4.6 and 2.1 kb), testis (4. 7 and 3.5 kb) and germ cells (4.5 and 3.5 kb). In addition, a third transcript was detected in rat pituitary (1.9 kb) and in rat testis and germinal cells (2.1 kb). The present study demonstrates that GnRH-R mRNA is expressed in rat and mouse testicular germ cells. We suggest that GnRH-R present in these cells may interact with GnRH or GnRH-like peptides produced in the testis and may be part of a paracrine system. The presence of multiple GnRH-R encoding transcripts is also of interest and warrants further studies to evaluate their regulation and function.

Poly(ADPribosyl)ation System in Rat Germinal Cells at Different Stages of Differentiation

In order to study the possible functional relationship between poly(ADP-ribosyl)ation and spermatogenesis, the three main germinal cell types have been isolated and characterized as haploid spermatids and diploid and tetraploid spermatocytes. Purified germinal cell populations and rats of different age were used for activity-, immuno-, and Northern blot experiments, to determine at which level poly(ADPR)polymerase (PARP) is regulated at various stages of spermatogenesis. Poly(ADPR)glycohydrolase (PARG) activity was also determined, as was the subcellular distribution of both PARP and PARG enzymes. The results show that the maximum of both PARP amount and PARP activity can be detected on tetraploid spermatocytes which undergo meiotic division, whereas PARG activity does not differ in germinal cells; the cytoplasmic form of this enzyme is prevalent in testis. Moreover, a difference in timing was observed in maximal level between PARP expression, determined on testis from 60-day-old rats, and PARP activity, detected on testis from 30-day-old animals. It seems that different mechanisms modulate the poly(ADPribosyl)ation system during spermatogenesis. Regulation of the poly(ADPribose) turnover, variations of PARP amount, as well as changes of PARP transcription level, seem to accompany germinal cell differentiation, possibly being implicated in DNA replication, repair, and transcription.

Three structures associated with the nucleolus in male rat germinal cells: Round body, coiled body, and “nubecula” and general presence of round body at male meiosis

In addition to chromosomes and nucleoli, three structures, i.e., round body, coiled body, and nubecula, are encountered in the nucleus during the meiotic prophase in male rats. These structures have been examined by electron microscopy in random and serial sections. The round body is a finely fibrillar, proteinaceous structure closely associated with the granular component of a nucleolus in rat spermatocytes and young spermatids. A similar structure has been observed in man, the monkey Macaca mulatta, the gastropod Achatina fulica, and the insect Locusta migratoria. Together with evidence from the literature, these results support the view that the round body is of general occurrence in the male meiocytes of eukaryotes and may, therefore, play a role in meiosis. The coiled body is a group of electron-dense elements called "coils", which average 35 nm in width, except after mid-pachytene when their size almost doubles. The coils are composed of 2-nm-wide filaments and 8 to 10-nm-wide granules, both of which are ribonucleoprotein. The coiled bodies are interpreted to be groups of "spliceosomes", that is, structures containing heterogeneous RNA and small nuclear RNA. A remarkable feature of the coiled body is its temporary disappearance at early pachytene and its reappearance at late pachytene, possibly due to drastic changes in the turnover rate of its component RNAs. The nubecula is a newly identified nuclear inclusion, composed of weakly staining threads loosely organized into a 560 nm-wide spheroid. It has been observed only in early pachytene nuclei.

Uptake and metabolism of retinol in isolated cells of germinal epithelium in vitro.

Retinyl esters were quantitatively the most significant product formed in short term incubations of isolated rat germinal cells administered 3H-retinol. 3H-retinyl palmitate was found to be the single most abundant metabolite accounting for approximately 80% of the total label 2 h after administration of 3H-retinol bound to bovine serum albumin. Differences were found in the relative activity of retinol uptake and metabolism between the various subpopulations of germinal cells separated by the staput sedimentation technique. The strongest correlation was between the magnitude of 3H-retinyl palmitate synthesis and the distribution of round spermatids. Large pachytene spermatocytes also appeared to correlate with 3H-retinyl palmitate synthesis although this correlation was ambiguous due to the presence of a small population of Sertoli cells and spermatogonia in these fractions. The concentration of cellular 3H-retinol was more than 10 fold greater in germinal cells administered 3H-retinol bound to bovine serum albumin than when 3H-retinol bound to serum retinol-binding protein was administered, and likewise the concentration of cellular 3H-retinyl palmitate was approximately 30 fold higher. In comparison to germinal cells, Sertoli cells administered 3H-retinol bound to serum retinol-binding protein incorporated 20 fold more 3H-retinol and synthesized more 3H-retinyl palmitate. However, both germinal cells and Sertoli cells were found to have incorporated similar concentrations of total label when administered 3H-retinol bound to bovine serum albumin. (ABSTRACT TRUNCATED AT 250 WORDS)

Action of gossypol on rat germinal cells. II. The acrosome.

The action of gossypol on the acrosomal complex in rats has been investigated by microscopical and submicroscopical methods. The drug displays its morphological action only on spermatozoa during the transit through the epididymis, causing malformations and vesiculations. It appears to exert a primary action on the S-S groups formation, disturbing morphological molding occurring in this period and inhibiting capacitation.

The action of gossypol on rat germinal cells

AbstractAdult male Sprague‐Dawley rats were fed daily with 25 mg/kg of gossypol acetic acid for 55 days. The examination of the testis revealed the constant integrity of the blood‐testis barrier. The earliest damage to the germinal cells appeared on the 14th day of treatment in spermatids in stage 18‐19, and consisted of mitochondrial swelling and cristae disorganization, which was manifested in an altered assembly of the mitochondrial helix and possible axonemal alterations. It appeared that the target cell was a 30–37‐day‐old spermatid that showed the first alterations 6–14 days later, when it reached stage 18. The mitochondrial and eventual axonemal damage was conserved after the migration into the epididymis, affecting 6% of the total sperm population on the 14th day of treatment and reaching the maximum (100%) on the 25th day of treatment. On the 5th day of treatment, epididymal spermatozoa revealed, moreover, the presence of other tail alterations: lack of half of the axonemal components (also in sperm with unaffected mitochondria), loss of midpiece plasma membrane, and breaking of the outer accessory fibers. The conclusion is that a direct action of gossypol on epididymal spermatozoa is superimposed on the action earlier exerted on the spermatids.

Effect of essential fatty acid deficiency on lipids of rat Sertoli and germinal cells.

The effect of essential fatty acid deficiency on lipids of separated rat Sertoli and germinal cells was determined at various time intervals after the animals were placed on a fat-free diet. Alterations in the fatty acid composition typical of essential fatty acid deficiency were noted in the Sertoli cells as well as in germinal cells as early as Days 9-14 on the fat-free diet. These changes included increased concentrations of oleic acid (18:1W9)2 and the appearance of 20:3W9. In some of the rats there were also decreases in linoleic (18:2W6) and arachidonic acids ((20:4W6), but no significant differences were found between Sertoli and germinal cells. Feeding a corn oil diet to rats previously maintained on a fat-free diet for 4 weeks reversed the changes in fatty acid composition of both Sertoli and germinal cells at the times studied. Lipid changes in phospholipids closely reflected those observed in the total lipids. These early changes in Sertoli cell lipids caused by an essential fatty acid deficiency may have important consequences since the Sertoli cell lipids caused by an essential fatty acid deficiency may have important consequences since the Sertoli cells play a significant role in the spermatogenic process.

The mutagenic potential of ethylene oxide using the dominant-lethal assay in rats

The mutagenicity of ethylene oxide in mammalian germinal cell lines was investigated using the dominant-lethal test in rats. Male rats were exposed to 1000 ppm of ethylene oxide for 4 hr and mated each week to groups of two females for 10 weeks. A significant increase in postimplantational fetal deaths was noted in the test group when compared to the negative control group. This significant increase was noted only during the first 5 weeks of the experiment, corresponding to the residence time of germinal cells exposed to ethylene oxide after their meiotic division. Triethylenemelamine served as the positive control. These results indicate that ethylene oxide is capable of inducing mutagenic changes in rat germinal cells.