Sperm aneuploidy and recurrent pregnancy loss.

Experiments of double target in-situ hybridization were performed separately for chromosomes 1-17, 8-18 and sex chromosomes on sperm samples from 20 couples suffering from three or more recurrent first trimester abortions. For a subset of this study population, additional experiments of multicolour fluorescence in-situ hybridization for chromosomes 4, 7, 12, 13, 15, 18, 21, and 22, were performed on the bases of the available data from abortive tissue karyotyping. A markedly high rate of sperm disomy (14.5-15.5%) was scored in only two cases. For three other patients, the cumulative disomy rates for chromosomes 1, 17, 8, 18, X and Y also increased but at a lower level (7.8-9.5%). For the remaining 15 patients, the frequency of sperm aneuploidy was moderately increased or normal. Men with recurrent pregnancy loss (RPL) and poor semen quality had baseline sperm aneuploidy and diploidy rates higher than men with normal semen parameters (with or without RPL). Using probes for chromosomes 1, 17, 8, 18, X and Y, significantly elevated frequencies of sperm aneuploidy (not diploidy) were found in 10% of men with a history of RPL. Their rate of sperm aneuploidy was 30-34%. For the other men, changes in sperm aneuploidy were not thought to affect RPL. Poor semen quality per se impacted negatively on sperm aneuploidy and diploidy, thus making the interpretation of clinical data more difficult.

Prolactin effect on pre-pubertal Sertoli cell proliferation and metabolism.

Direct effects of PRL on Sertoli cell proliferation were investigated by using Sertoli cell primary cultures isolated from both prepubertal rat and porcine testes. PRL metabolic effects were analyzed in rat Sertoli cell primary cultures. Exposure to physiological doses of PRL resulted in a significant increase (+50-60%) of basal DNA synthesis, as reflected by the pattern of [3H] thymidine incorporation during culture; significant increases in lactate secretion (about 50%), androgen binding protein (ABP) production (about 30%) and basal protein synthesis (25-30%), as reflected in the augmented [14C] valine incorporation, were also evident. Taken together, our present findings, indicating significant effects of PRL on Sertoli cell proliferation and metabolism, demonstrate that Sertoli cells are a potential target for PRL action at testicular level during pre-pubertal development.

Regulation of renal growth and IGFBP-4 expression by triiodothyronine during rat development.

The insulin-like growth factors (IGFs) and insulin-like growth factor binding proteins (IGFBPs), which regulate IGF activity, play a fundamental role in renal cell proliferation and differentiation. The thyroid hormone is considered to be required for kidney development; excess induces local hypertrophy and hyperplasia. The aim of the present study was to investigate the possible involvement of the IGF/IGFBP system in thyroid hormone-induced renal growth during the development of the rat. Our results show that thyroid hormone withdrawal by 6-propyl-2-thiouracil (PTU)-treatment of rats at all ages had no effect on renal IGFBP-4 mRNA levels, whereas the abundance of the serum protein was decreased compared to controls. Intraperitoneal triiodothyronine (T3) administration to hypothyroid rats resulted in renal hypertrophy associated with a significant upregulation of IGFBP-4 expression with increased levels of renal IGFBP-4 mRNA and serum protein. T3-induced upregulation of IGFBP-4 expression suggests the involvement of the local IGF/IGFBP system in T3-induced renal hypertrophy.

Metabolic effects of L-carnitine on prepubertal rat Sertoli cells.

The role of carnitine on Sertoli cell metabolism was investigated. Carnitine effects on Sertoli cell lipid metabolism were evaluated by measuring the intracellular levels of non-esterified fatty acids (NEFA) and ketone bodies. The concentration of NEFA in Sertoli cell cultured in the presence of carnitine is significantly reduced as compared to control, while, no significant changes were observed in the concentration of ketone bodies. The functional parameters evaluated to assess the influence of carnitine on Sertoli cell carbohydrate metabolism, i.e., lactate and pyruvate production, lactate dehydrogenase activity and hexose transport, were all significantly increased following carnitine in vitro supplementation. Thus, carnitine appears to drive Sertoli cell intermediary metabolism in an intimately interrelated way, stimulating both fatty acid breakdown and glycolysis. Our results indicate that Sertoli cells are a possible target for a widespread metabolic action of carnitine and strongly support the involvement of carnitine in the regulation of Sertoli cell functions which are related with germ cell "nutrition", convincingly suggesting a direct influence of the compound at testis level.

Thyroid hormone and retinoic acid induce the synthesis of insulin-like growth factor-binding protein-4 in prepubertal pig sertoli cells.

A large body of evidence suggests the existence of an intratesticular IGF system complete with ligands, receptors and binding proteins (IGFBPs); the aim of the present study was to evaluate tri-iodothyronine (T(3)) and retinoic acid (RA) effects on IGFBP production by Sertoli cells. A significant dose-dependent increase in IGFBP-4 mRNA levels was observed in Sertoli cells cultured in the presence of physiological concentrations of T(3) or RA. This response was inhibited by cycloheximide, indicating that de novo protein synthesis is required, as well as by actinomycin D, suggesting that the increase in mRNA levels requires transcriptional activation. As shown by ligand blot assays the stimulatory effects of both agents on IGFBP-4 mRNA expression appears to be consistent with an enhanced synthesis and secretion of IGFBP-4, thus suggesting that the transcriptional response is transduced to the protein level. Our data establish an important direct role for T(3) and RA in regulating IGFBP-4 expression and consequently IGF activity at the testis level.

IGF-I production by adult rat hepatocytes is stimulated by transforming growth factor-alpha and transforming growth factor-beta1.

Previously, we have observed that epidermal growth factor (EGF), a potent mitogen for cultured hepatocytes, stimulates the production of IGF-I and IGF-binding proteins (IGFBPs) by cultured hepatocytes from adult rats. This study was undertaken to investigate the possibility that other growth factors of hepatic origin could specifically be involved in the regulation of IGF-I and IGFBP expression. The effects of transforming growth factor-alpha (TGF-alpha), through EGF receptors to induce a mitogenic response, and transforming growth factor-beta1 (TGF-beta1), produced by non-parenchymal liver cells and able to inhibit hepatocyte proliferation in vivo and in culture, have been studied in cultured adult rat hepatocytes. Our results demonstrate that TGF-alpha and TGF-beta1 significantly stimulate IGF-I and IGFBP secretion by cultured hepatocytes but no change in the abundance of IGF-I and IGFBP mRNAs was observed with respect to controls. Cycloheximide is able to inhibit both basal and TGF-stimulated release of IGF-I and a similar effect was elicited by octreotide, the somatostatin analog, known to directly affect hepatic IGF-I gene expression. Our findings show the role of the liver in the secretion of IGF-I and IGFBPs, not only under endocrine and nutritional control but also under autocrine and paracrine control.

Tri-iodothyronine increases insulin-like growth factor binding protein-2 expression in cultured hepatocytes from hypothyroid rats.

Previous evidence suggests the existence of a thyroid hormone-IGF axis in the liver and changes in hepatic insulin-like growth factor binding protein (IGFBP) expression in rats with altered thyroid status have been previously reported. The aim of this study was to check if the higher IGFBP-2 mRNA levels observed in liver of hypothyroid rats could be due to a direct effect of thyroid hormone on the IGFBP-2 gene. In our experiments, cultured hepatocytes isolated from normal and hypothyroid adult rats were used. Northern blot analysis revealed barely detectable IGFBP-2 mRNA in normal rat hepatocytes, but easily detectable signal in hypothyroid rat cells. Therefore, the effect of tri-iodothyronine (T3) was investigated using cultured hepatocytes from hypothyroid rats as an in vitro model. The IGFBP-2 message was increased in a dose-dependent manner in hepatocytes cultured for 12-24 h in the presence of T3. A similar increase occurred in accumulation of IGFBP-2 in the culture medium, as measured by RIA. The effect of T3 on IGFBP-2 transcript levels appeared to consist of enhanced gene transcription and was independent of ongoing protein synthesis, but it was completely abolished by the incubation of hepatocytes with insulin. The latter result confirmed the dominant role of insulin in regulating IGFBP-2 expression by cultured hepatocytes. In vivo experiments confirmed an increase in hepatic IGFBP-2 mRNA and serum IGFBP-2 levels in hypothyroid rats and demonstrated, in addition, a significant increase in these measures in T3-treated rats. Taken together, our in vitro and in vivo results support a role for a thyroid hormone-IGF axis in the liver and suggest that other factors, such as insulin, interact in vivo with thryoid hormone in regulating hepatic IGFBP-2 expression.

Poly(ADP-ribose) polymerase activity in regenerating liver of hypothyroid rats.

The role of PARP, a nuclear enzyme involved in DNA synthesis, repair and cell transformation, was studies during liver regeneration in hypothyroid animals. Hypothyroidism was induced by in vivo administration of propylthiouracil. In regenerating euthyroid animals PARP activity is stimulated showing an early and significant increase at 1.5 h with a maximum at 6 h after partial hepatectomy. Such an increase returns to control values within 18 h preceding the onset of DNA synthesis. A markedly different behavior, with respect to euthyroids, has been evidenced in hypothyroid rats. At first, liver PARP level was about 2-fold higher in non regenerating hypothyroid rats with respect to control euthyroids. During regeneration, PTU-treated animals show a net decrease in PARP activity, with a minimum at 6-9 h after partial hepatectomy. The activity returns to control levels within 24 days. The minimum in PARP activity anticipates, also in this case, the onset of DNA synthesis, which exhibits a maximum at 15-18 h. During liver regeneration PARP activity shows modifications related to the beginning of de novo DNA synthesis. Furthermore, these variations in turn undergo the effects of hypothyroidism.

DNA topoisomerase I activity in regenerating liver of hypothyroid rats.

DNA topoisomerase I activity is known to be inhibited by poly(ADP-ribosyl)ation. Both poly(ADP-ribose)polymerase and DNA topoisomerase I participate to major biological events, such as DNA transcription, repair and synthesis. Previously, a 2-fold increase in PARP activity has been shown in hypothyroid animals. Using the regenerating rat liver model, we have studied the behaviour of DNA topoisomerase I activity in hypothyroid rats. PARP activity, was also studied in another set of experiments. DNA topoisomerase I relaxing activity was determined on supercoiled plasmid DNA, and topoisomers separated by agarose gel electrophoresis. An increase in the relaxing activity of Topo I was observed early after hepatectomy. This enhancement well correlates with the reported inhibition of PARP activity at the scheduled times. The data from hypothyroid animals support an inverse relationship between PARP and Topo I. These results are completely reversed with respect to those obtained during liver regeneration in euthyroids.

Tri-iodothyronine increases insulin-like growth factor binding protein-4 expression in rat hepatocytes.

Previous in vivo studies demonstrated significant variations in insulin-like growth factor binding protein-1 (IGFBP-1), IGFBP-2 and IGFBP-4 hepatic mRNAs and/or serum levels depending on the rat thyroid status. In this study we employed cultured hepatocytes from adult rats to demonstrate a possible direct regulation of these genes by tri-iodothyronine (T3). Northern blot analysis revealed that IGFBP-1 and -4 messages were clearly expressed, whereas IGFBP-2 signal was barely detectable. No significant effects on IGFBP-1 mRNA level or on peptide secretion were detected in T3-cultured hepatocytes. In contrast, significant increases in IGFBP-4 mRNA steady-state levels as well as in IGFBP-4 secretion were observed in hepatocytes cultured for 12-24 h in the presence of T3. The T3 effect on IGFBP-4 transcript levels appears to consist of enhanced gene transcription and is independent of ongoing protein synthesis. The T3-increased IGFBP-4 expression in cultured hepatocytes is consistent with our in vivo experiments demonstrating an increase in hepatic IGFBP-4 mRNA and serum IGFBP-4 levels in T3-treated rats. Furthermore, significant decreases in hepatic IGFBP-4 message and serum IGFBP-4 levels were observed in hypothyroid rats compared with euthyroid controls. Our data establish an important direct role for thyroid hormone in regulating IGFBP-4 expression and consequently IGF activity.

Regulation of IGFBP-1 and -4 expression by triiodothyronine (T3) in cultured hepatocytes is cell- and gene-specific.

Evidence suggests that thyroid hormone plays a role in the regulation of hepatic IGF/IGFBP expression both in human and rats. In this study we compared the effect of T3 on IGFBP-1 and -4 expression in rat hepatocyte primary cultures and in the human hepatoma cell line HepG2. Northern blot analysis revealed that IGFBP-1 mRNA levels were not affected by T3 in cultured rat hepatocytes, whereas a net increase of IGFBP-1 transcript abundance was induced by the hormone in HepG2 cells. On the contrary, IGFBP-4 mRNA levels were increased in rat hepatocytes cultured in the presence of T3, but unaffected in T3-treated HepG2 cells. Therefore, thyroid hormone seems to regulate hepatic IGFBP expression in a direct and gene-specific way. Moreover, the effects of thyroid hormone depend strictly on the source of target hepatocyte.

Thyroid hormone affects rat uterine expression of IGF-I and IGFBP-4.

The rat uterus has been shown to be a site of production of insulin-like growth factor-I (IGF-I) and multiple IGF-binding proteins (IGFBP-2, -3, -4, -5, -6) which are involved in estrogen-induced uterine proliferation. The presence of T3-receptors in rat uterus suggests a role of thyroid hormone in the regulation of uterus responses to estradiol. In this study IGF-I and IGFBP-4 mRNAs in uterus, oviduct and cervix from euthyroid, hypothyroid and T3-treated rats were quantified by Northern blot analysis. Our results demonstrate: i) a marked decrease in IGF-I and IGFBP-4 mRNA levels in the uterus but an increase in the oviduct of hypothyroid rats; ii) a marked increase in IGF-I and IGFBP-4 mRNA levels in the uterus but a net decrease in the cervix of T3-treated rats. The uterine changes in IGF-I and IGFBP-4 mRNA levels associated with hypothyroid status were in agreement with those observed in liver.

Relationship between DNA synthesis and growth factor expression in primary cultures of adult rat hepatocytes.

In this study we employed primary culture of adult rat hepatocytes to verify the effects of two different extracellular matrices (collagen, matrigel) on EGF-stimulated DNA synthesis and c-myc expression. Our results confirm that in adult rat hepatocytes EGF induces DNA synthesis, preceded by a transient increase of c-myc expression, when cells are cultured at low density on collagen. DNA synthesis appears to be in reciprocal relationship with hepatic expression of IGF-I, IGFBP-1, IGFBP-2 and IGFBP-4, suggesting that IGF-I/IGFBPs system is not involved in liver growth.

Triiodothyronine decreases gammaglutamyltranspeptidase expression in cultured rat hepatocytes.

Recently, an increase in gammaglutamyltranspeptidase (GGT) activity and mRNA in liver of hypothyroid rats has been reported. The aim of this study was to verify if triiodothyronine (T3) can directly affect GGT expression in primary cultures of rat hepatocytes. Results obtained from adult rat hepatocytes cultured in serum-free medium demonstrate: 1) a rise in GGT mRNA level magnified by dexamethasone during the maintenance of hepatocytes in culture which parallels the stimulation of GGT activity; 2) a negative effect of T3 on GGT activity of cultured hepatocytes which reflects a specific inhibition of GGT gene expression. The T3 effect on GGT expression in cultured hepatocytes is in line with previous observations on hypothyroid rat liver suggesting an important role for thyroid hormone in maintaining the differentiated adult liver phenotype in the rat.