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R M Sharpe

Publications and source records attributed to R M Sharpe.

At least 55 records · Page 3Linked to original sources
PubMed1997

Expression cloning of a rat testicular transcript abundant in germ cells, which contains two leucine zipper motifs.

The aim of the present study was to identify specific, novel germ cell markers that could be used to monitor normal and abnormal spermatogenesis. Of several cloned cDNAs isolated from an adult rat testis cDNA library using an expression screening strategy, clone 813B4 (700 base pairs) hybridized exclusively to three mRNA transcripts in samples isolated from rat testes on and after Day 21 of life and to epididymides from some, but not all, adult rats. After further screening, two identical clones encoding a 2.2-kilobase cDNA (KTT4) were isolated and found to contain an open reading frame of 578 amino acids including two leucine zipper motifs. On Northern blots, KTT4 mRNA was abundant in samples from round spermatids, and homologous mRNAs were present in testes from mice and marmosets. A zoo blot revealed that the KTT4 gene is conserved in humans, monkeys, mice, dogs, and cattle. On sections of rat testes, KTT4 mRNA was first detectable in pachytene spermatocytes at stage VII and thereafter was abundant in round and elongating spermatids until step 15. Expression of KTT4 was not altered by ethane dimethane sulphonate-induced androgen withdrawal, but in rats treated 14 days previously with methoxyacetic acid, a marked reduction in KTT4 was noted associated with the depletion of round spermatids. In conclusion, the present study identified a conserved gene expressed in meiotic and post-meiotic germ cells; database searches have shown it to be homologous to recently published sequences for an outer dense fiber protein of the sperm tail (Odf2/Odf84).

Amino Acid Sequence↗
PubMed1997

Testicular expression of inhibin and activin subunits and follistatin in the rat and human fetus and neonate and during postnatal development in the rat.

Inhibins, activins, and follistatins are all believed to play roles in the regulation of FSH secretion by the pituitary and in the paracrine regulation of testis function. Previous studies have resulted in conflicting data on the pattern of expression of the inhibin/activin subunits, and little information on expression of follistatin during fetal/neonatal life. We have made use of new, highly specific monoclonal antibodies and fixed tissue sections from fetal, neonatal, and adult rats, and limited amounts of fetal and neonatal human testis, to undertake a detailed immunocytochemical study of the pattern of expression of these regulatory proteins. In the rat, positive immunostaining for the alpha-subunit of inhibin (alpha) was first detectable on day 14.5 post coitum (p.c.), the first day on which the testis could be morphologically distinguished from the ovary. During fetal life, the alpha-immunostaining was most prominent in the fetal Leydig cells. In Sertoli cells, alpha-immunostaining was slightly stronger on days 14.5 and 15.5 p.c. compared with 16.5-20.5. After birth, alpha-immunostaining remained intense in fetal Leydig cells but declined following their replacement with their adult-type counterparts; in contrast, alpha-subunit increased in Sertoli cells immediately after birth. Immunostaining with antibodies specific to betaB-subunit showed a similar pattern to that of the alpha-subunit, except that positive immunostaining was first detectable on day 16.5 p.c., 2 days later than immunostaining for the alpha-subunit. The pattern of betaB-immunostaining in postnatal samples paralleled that of the alpha-subunit. Immunostaining using antibodies against the betaA-subunit did not produce any significant reaction product in any sample. Follistatin was undetectable in the fetal rat testis but appeared in the Leydig cells immediately after birth and its expression remained intense throughout postnatal development and in adult testis. No evidence was obtained for expression of either the inhibin/activin subunits or follistatin in the germ cells, peritubular myoid cells, or other interstitial cells in any of the sections examined. In the human fetal testis, both alpha- and betaB-subunits were immunodetectable at 16, 18, and 24 weeks gestation in Sertoli and Leydig cells, with stronger immunostaining in Sertoli cells at 24 weeks. Postnatally at 4 months, immunoexpression of the betaB-subunit was no longer detectable, whereas the alpha-immunostaining became weaker but was still present in both Sertoli and Leydig cells. No positive immunostaining for betaA-subunit or follistatin was detectable at any time point studied. In conclusion, we have shown that, in the rat testis, the majority of inhibin alpha-subunit and inhibin/activin betaB-subunit is immunolocalized to the fetal-type Leydig cells during fetal/neonatal life but, following birth, immunoexpression in the Sertoli cells of both subunits increases markedly while follistatin is immunodetectable only postnatally.

Activins↗
PubMed1997

Environmental oestrogens--present understanding.

Three years ago it was hypothesized that the reported adverse changes in male reproductive health could be explained by exposure to compounds with oestrogenic (or other hormone disruptive) activity. Although this issue has been highly publicized, there has been little progress towards a realistic assessment of whether environmental oestrogens pose a health risk to humans. This article attempts to give a brief overview of the current status of knowledge concerning environmental oestrogens and highlights some of the difficulties associated with risk assessment. Compounds within several major groups of chemicals, including organochlorine pesticides, polychlorinated biphenyls, phenolic compounds and phthalate esters, have been identified as being weakly oestrogenic by in vitro and in vivo screening methods. Many of these compounds are widespread and persistent in the environment. They are likely to be present in the food chain, drinking water, plastics, household products and food packaging, although which is the most important route of human exposure is unclear. In addition to exposure to man-made chemicals, the consumption of plant-derived oestrogens in foodstuffs poses a potential risk to human health as phytoestrogens are more potent oestrogens and their intake by some infants is likely to be considerable.

Animals↗
PubMed1997

Investigation of the potential role of the germ cell complement in control of the expression of transferrin mRNA in the prepubertal and adult rat testis.

Iron is required for the normal development of germ cells during spermatogenesis. Because these cells have no direct access to systemic iron, there exists a shuttle system involving production and secretion of the iron-transporting protein transferrin by the Sertoli cells. Previous reports using cultures of immature Sertoli cells exposed to adult germ cells, or in vivo studies involving germ cell depleted adult rat testes, concluded that production of transferrin by Sertoli cells is modulated by germ cell complement. In the present study we have used in situ hybridisation with cRNA probes directed against the 5' and 3' ends of transferrin mRNA to examine the pattern of expression of transferrin in the immature and adult rat testis. Adult rats were treated with ethane dimethane sulphonate or methoxyacetic acid (MAA) to manipulate their testosterone levels or germ cell complement respectively. Initial findings obtained using the 3' probe showed a decrease in transferrin mRNA associated with round spermatid depletion. However, these data were not confirmed by in situ hybridisation when the 5' probe was used. The specificity of the probes was examined using Northern blotting and the 3' probe was found to hybridise to the germ cell transcript for hemiferrin even under conditions of high stringency. Examination of immature and pubertal rat testes by in situ hybridisation using the 5' transferrin-specific probe found that as early as 14 days of age the level of expression of transferrin mRNA was clearly different between tubules, and the mRNA appeared to be expressed in Leydig cells on and after day 31. In the adult rat testis, maximal expression of transferrin mRNA was found at stages VIII-XIV, calling into question the interpretation of the results of some previous studies showing expression of transferrin mRNA at all stages of the spermatogenic cycle. This stage-specific pattern of expression was not altered by acute germ cell depletion using MAA. However, Northern blot analysis showed a statistically significant increase in transferrin mRNA expression at 7 days after MAA treatment when pachytene spermatocytes were depleted from tubules at all stages of the spermatogenic cycle at which transferrin is normally expressed. In conclusion, we found that transferrin mRNA expression was not modulated by round spermatids as has been reported previously but that meiotic germ cells may influence expression of transferrin at specific stages of the spermatogenic cycle.

Animals↗
PubMed1997

Immunolocalisation of oestrogen receptor-alpha within the testis and excurrent ducts of the rat and marmoset monkey from perinatal life to adulthood.

The sites of action and the physiological role of oestrogens in the male reproductive tract are poorly understood. We have undertaken a systematic study of the immunoexpression of oestrogen receptor-alpha (ER alpha) in the male rat from late fetal life through to adulthood and compared the findings with results obtained in the marmoset monkey (Callithrix jacchus) from neonatal to adult life. The testes, rete testis, efferent ducts and epididymis were examined from normal male rats (aged 4, 8, 10, 15, 20, 25, 38, 48 and 90 days) and from male rat fetuses on days 17.5 and 18.5 of gestation; comparable tissues were examined from neonatal, infantile, peripubertal and adult marmosets aged 8, 18-24, 54-62 and 92-112 weeks respectively. Immunolocalisation of ER alpha used antigen retrieval and a monoclonal antibody directed to the N-terminus, which had proved superior to six other antisera tested. ER alpha was immunoexpressed in interstitial cells, including the fetal/ neonatal generation of Leydig cells, in both the rat and marmoset. In the rat, the adult generation of Leydig cells were also immunopositive for ER alpha whereas the comparable cells in the marmoset were only weakly immunopositive. ER alpha was not expressed in Sertoli cells, peritubular myoid cells, blood vessels or germ cells at any time in either species. In late fetal life in the rat, ER alpha was immunoexpressed in cells surrounding the mesonephric tubules, whereas postnatally it was expressed in the epithelium of the rete testis and efferent ducts at all ages from 4 to 90 days; this immunoexpression was most pronounced in the efferent ducts. In the marmoset, the efferent ducts, but not the rete testis, also showed intense immunoexpression of ER alpha. Apart from sporadic immunostaining for ER alpha in the epididymal duct of the rat in the neonatal period, the caput, corpus and cauda epididymis were negative for immunoexpression of ER alpha at all ages in both species. These findings suggest that the main actions of oestrogens in the male reproductive tract, mediated by ER alpha, are related to the development and function of the efferent ducts and the Leydig cells. In consideration of data from this and previous studies of oestrogen binding, we predict possible sites of expression of other oestrogen receptors (e.g. ER beta) in Sertoli cells and the epididymis. Interactive effects, related to the relative levels of androgens and oestrogens, could be physiologically important in the excurrent ducts of the adult testis.

Age Factors↗
PubMed1997

Regulation of the secretion and synthesis of rat Sertoli cell SGP-1, SGP-2 and CP-2 by elongate spermatids.

The aim of this study was to investigate the effect of the absence of elongate spermatids (ES) from the rat seminiferous epithelium on the quantitative secretion and synthesis of the three major Sertoli cell secretory proteins--SGP-1, SGP-2 and CP-2. Seminiferous tubules (ST) were isolated (a) from normal 28-day-old rats, in which the most mature germ cell type is the round spermatid, (b) from normal adult rats at stages IX-XIV of the spermatogenic cycle, i.e. after spermiation, or at stages I-V and VI-VIII, when ES are still attached to the Sertoli cell, and (c) at stages VI-VIII from normal adult rats and from rats treated with methoxyacetic acid (MAA) in order to specifically deplete ES at these stages. Two-dimensional SDS PAGE combined with computerized image analysis was used to analyse 35S-methionine-labelled intracellular and secreted proteins. In the case of SGP-1 and SGP-2, almost all of the protein synthesized by ST was secreted. The total amount of both SGP-1 and CP-2 secreted by unstaged ST from immature rats was significantly lower than that secreted by unstaged ST from adult rats. The total amount of SGP-1 and CP-2 secreted by adult ST at stages IX-XIV of the spermatogenic cycle also declined dramatically compared to ST at earlier stages. The proportion of the total CP-2 synthesized by ST which was secreted also declined in all situations in which ES were absent from the seminiferous epithelium. The synthesis of only SGP-2 was changed by ES depletion from ST at stages VI-VIII, which was almost doubled compared to synthesis of this protein by ST from control rats. Our results suggest strongly that the secretion of SGP-1 and SGP-2 is via the constitutive pathway, and that regulation of these two proteins by ES is at the level of protein synthesis. In contrast, the regulation of CP-2 by ES is predominantly at the level of secretion, suggesting that this protein is secreted via a regulated pathway. Our findings add to the evidence showing that ES play a major role in the regulation of Sertoli cell function.

Acetates↗
PubMed1996

Expression of cytochrome P450 17alpha-hydroxylase/C17-20 lyase in the fetal rat testis is reduced by maternal exposure to exogenous estrogens.

Testosterone is required for normal development of the male reproductive tract. Synthesis of testosterone occurs in the Leydig cells and is dependent upon the expression of several enzymes, including cytochrome P450 17alpha-hydroxylase/C17-20-lyase (P450c17), which is highly regulated within the testis. The aim of the present study was to investigate whether maternal exposure to estrogenic chemicals was able to affect Leydig cell function in the developing male fetus at the time of masculinization. Pregnant rats were injected sc with diethylstilbestrol (DES; 100 or 500 micrograms/kg), 4-octylphenol (OP; 100 or 600 mg/kg), or vehicle (oil, control) on days 11.5 and 15.5 postcoitum. Doses were chosen to reflect the reported estrogenic potency of the chemicals in vitro. On day 17.5, fetal testes were fixed before performing in situ hybridization and immunocytochemistry, used for extraction of RNA, or homogenized in phosphate buffer for determination of 17alpha-hydroxylase enzyme activity. There was no difference between fetuses from control and treated mothers in either the overall histology of the testes or the apparent number of Leydig cells, as determined by immunocytochemistry with an antibody directed against 3beta-hydroxysteroid dehydrogenase. However, there was a consistent and striking reduction in the amount of P450c17 detected by immunocytochemistry in testes from the groups given the higher dose of DES or OP. These observations were supported by measurement of 17alpha-hydroxylase activity, which was significantly reduced compared with that in controls (6.25 +/- 0.65 pmol/testis x min) in fetuses from animals treated with 100 micrograms/kg DES (4.27 +/- 0.39; P < 0.05), 500 micrograms/kg DES (1.4 +/- 0.47; P < 0.001), or 600 micrograms/kg OP (4.25 +/- 0.33; P < 0.05). RT-PCR and in situ hybridization revealed that these changes were mirrored by reductions in P450c17 messenger RNA in testes from fetuses from treated mothers compared with control levels. In conclusion, maternal treatment with either a potent sythetic estrogen (DES) or a putative environmental estrogen (OP) results in reduced expression of the messenger RNA and protein for P450c17 in fetal Leydig cells. These results, therefore, provide a mechanism by which inappropriate exposure of the fetus to estrogenic chemicals might have an adverse effect on fetal steroid synthesis and masculinization.

Aldehyde-Lyases↗
PubMed1995

Colocalization of mRNA and protein using in situ hybridization and immunohistochemistry in testicular tissue.

Using testes fixed by perfusion with Bouin's fluid and embedded in paraffin wax, this study has established methods for combining in situ hybridization and immunocytochemistry on the same section to colocalize mRNA and protein for transition protein-1 (TP-1) and sulfated glycoprotein-1 (SGP-1), respectively. It was found that SGP-1 could be detected in tissue sections subsequent to the detection of TP-1 mRNA in situ. The finding that 1) the tissue pretreatments required to permeabilize the section and to allow access to the probe, and 2) the hybridization conditions themselves, had no adverse effect on the detection of antigen, eases the performance of this technique. On this basis, important information could be obtained on the transcriptional and translational activity of spermatogenic cells, if related probes and antibodies are utilized.

Animals↗
PubMed1995

Phosphatidylethanolamine binding protein is an abundant secretory product of haploid testicular germ cells in the rat.

An abundant cellular and secretory product of isolated seminiferous tubules from adult rats was identified as having an apparent molecular weight of approximately 24,000 and a pI of 5.3 on autoradiographs of two-dimensional polyacrylamide gels. A protein with identical migration characteristics was identified as a major secretory product of isolated round spermatids. Microsequencing revealed that the protein had homology to phosphatidylethanolamine binding protein (PEBP) identified in rat brain. Primers were used in conjunction with RTPCR to amplify a partial cDNA which was used to probe a rat testis library to obtain full length clones. On Northern blots, PEBP mRNA was abundant in adult rat testis and epididymis and fractions enriched in germ cells but was very low/absent from fetal or immature rat testis or adult rat Sertoli cells. In situ hybridisation identified that abundant mRNA was first detectable in pachytene spermatocytes at stage VII and thereafter at particularly high levels in round and elongating spermatids until step 14. Proteins with significant sequence homology to the rat testis PEBP have been identified previously in mouse testis and epididymis, in rat germ cell cultures and coating the surface of mature rat sperm. Differences in the timing of expression of the PEBP mRNA (first expressed in pachytene spermatocytes) and secretion of the PEBP protein (not a major secretory product until round spermatids) is consistent with PEBP mRNA undergoing delayed translation. The role(s) of secreted lipid binding proteins in spermatogenesis are discussed.

Amino Acid Sequence↗
PubMed1995

Infertility in a transgenic rat due to impairment of cytoplasmic elimination and sperm release from the Sertoli cells.

In line TGR(mRen2)26 transgenic rats (TGR26) bearing a randomly inserted additional renin transgene, the males, but not the females, were found to be infertile. Tissue was obtained from TGR26 males and littermate controls after perfusion fixation, and the morphology of the testes and epididymides was examined. Testis size was normal as was gross morphology, but careful examination revealed that the release of many spermatozoa at stage IX of the spermatogenic cycle was impaired. In addition, the process of cytoplasmic elimination was abnormal, as cytoplasmic fragments of elongate spermatids were present in the epididymis. In TGR26 males, seminiferous tubule lumen size was significantly larger (p < 0.001) than in littermate controls, a difference that was most marked at stages IX-XIV--an effect that could be related to the retention of spermatozoa. In situ hybridization confirmed that expression of renin mRNA could be detected in testes of TGR26 rats but not in normal controls or in a fertile line (TGR27) of rats bearing the same transgene. Immunocytochemistry and in situ and Northern hybridization were used to elucidate the pattern of expression of genes that previous studies have implicated in the process of sperm maturation and/or release. Of the gene products examined (sulphated glycoproteins 1 and 2 [SGP-1, SGP-2], transition proteins 1 and 2 [TP-1, TP-2], urokinase, and cyclic protein 2[CP-2], none showed any major change in the pattern of expression compared with that in controls. We postulate that TGR26 transgenic male rats may be infertile because the expression of a gene (or genes) involved in the process of cytoplasmic elimination and/or sperm release has been disrupted by the presence of the transgene close to or within the gene(s). Future planned studies will involve determination of the insertion site(s) and ultrastructural analysis of the final phases of spermiogenesis.

Animals↗
PubMed1995

Infertility in a transgenic rat due to impairment of cytoplasmic elimination and sperm release from the Sertoli cells.

In line TGR(mRen2)26 transgenic rats (TGR26) bearing a randomly inserted additional renin transgene, the males, but not the females, were found to be infertile. Tissue was obtained from TGR26 males and littermate controls after perfusion fixation, and the morphology of the testes and epididymides was examined. Testis size was normal as was gross morphology, but careful examination revealed that the release of many spermatozoa at stage IX of the spermatogenic cycle was impaired. In addition, the process of cytoplasmic elimination was abnormal, as cytoplasmic fragments of elongate spermatids were present in the epididymis. In TGR26 males, seminiferous tubule lumen size was significantly larger (p < 0.001) than in littermate controls, a difference that was most marked at stages IX-XIV--an effect that could be related to the retention of spermatozoa. In situ hybridization confirmed that expression of renin mRNA could be detected in testes of TGR26 rats but not in normal controls or in a fertile line (TGR27) of rats bearing the same transgene. Immunocytochemistry and in situ and Northern hybridization were used to elucidate the pattern of expression of genes that previous studies have implicated in the process of sperm maturation and/or release. Of the gene products examined (sulphated glycoproteins 1 and 2 [SGP-1, SGP-2], transition proteins 1 and 2 [TP-1, TP-2], urokinase, and cyclic protein 2 [CP-2]), none showed any major change in the pattern of expression compared with that in controls. We postulate that TGR26 transgenic male rats may be infertile because the expression of a gene (or genes) involved in the process of cytoplasmic elimination and/or sperm release has been disrupted by the presence of the transgene close to or within the gene(s). Future planned studies will involve determination of the insertion site(s) and ultrastructural analysis of the final phases of spermiogenesis.

Animals↗
PubMed1995

Comparative analysis of proteins secreted in vitro by isolated seminiferous tubules from man and the rat.

The aims of this study were to provide an overall comparison of the in-vitro protein secretory profile of seminiferous tubules (ST) isolated from man and the rat, and to identify specific proteins secreted by both species. Two-dimensional SDS PAGE was used to compare the profile of proteins secreted by cultured ST from 15 men undergoing orchidectomy with those secreted by ST isolated at stages VI-VIII of the spermatogenic cycle from normal adult rats, and by ST at the same stages isolated from rats pretreated with methoxyacetic acid (MAA) in order to deplete both pachytene spermatocytes and round spermatids. Two abundant groups of proteins not present in the medium of rat ST were secreted consistently by human ST, though the profile of proteins secreted by human ST was otherwise more variable than in the rat. Twelve proteins secreted by ST isolated from humans were identified as possible homologues of rat ST-secreted proteins, including the major rat Sertoli cell products SGP-1 and SGP-2, and an androgen-regulated protein which derives in the rat from round spermatids. Otherwise, the majority of the 12 potential homologues corresponded to proteins which in the rat are secreted by ST from both normal and germ cell-depleted testes or by ST from germ cell-depleted testes only, suggesting that they are probably secreted by Sertoli cells and/or peritubular cells. Based on the potential homology of the proteins identified, our results suggest, first, that these proteins may play an important role in spermatogenesis and second, that the profile of proteins secreted by human ST is more akin to that secreted by ST isolated from germ cell-depleted rats.

Adult↗
PubMed1995

Gestational and lactational exposure of rats to xenoestrogens results in reduced testicular size and sperm production.

This study assessed whether exposure of male rats to two estrogenic, environmental chemicals, 4-octylphenol (OP) and butyl benzyl phthalate (BBP) during gestation or during the first 21 days of postnatal life, affected testicular size or spermatogenesis in adulthood (90-95 days of age). Chemicals were administered via the drinking water or concentrations of 10-1000 micrograms/l (OP) or 1000 micrograms/l (BBP), diethylstilbestrol (DES; 100 micrograms/l) and an octylphenol polyethoxylate (OPP; 1000 micrograms/l), which is a weak estrogen or nonestrogenic in vitro, were administered as presumptive positive and negative controls, respectively. Controls received the vehicle (ethanol) in tap water. In study 1, rats were treated from days 1-22 after births in studies 2 and 3, the mothers were treated for approximately 8-9 weeks, spanning a 2-week period before mating throughout gestation and 22 days after giving birth. With the exception of DES, treatment generally had no major adverse effect or body weight: in most instances, treated animals were heavier than controls at day 22 and at days 90-95. Exposure to OP, OPP, or BBP at a concentration of 1000 micrograms/1 resulted in a small (5-13%) but significant (p < 0.01 or p < 0.0001) reduction in mean testicular size in studies 2 and 3, an effect that was still evident when testicular weight was expressed relative to body, weight or kidney weight. The effect of OPP is attributed to its metabolism in vivo to OP. DES exposure caused similar reductions in testicular size but also caused reductions in body weight, kidney weight, and litter size. Ventral prostate weight was reduced significantly in DES-treated rats and to minor extent in OP-treated rats. Comparable but more minor effects of treatment with DES or OP on testicular size were observed in study 1. None of the treatments had any adverse effect on testicular morphology or on the cross-sectional area of the lumen or seminiferous epithelium at stages VII-VIII of the spermatogenic cycle, but DES, OP, and BBP caused reductions of 10-21% (p < 0.05 to p < 0.001) in daily sperm production. Humans are exposed to phthalates, such as BBP, and to alkylphenol polyethoxylates, such as OP, but to what extent is unknown. More detailed studies are warranted to assess the possible risk to the development of the human testis from exposure to these and other environmental estrogens.

Animals↗
PubMed1995

Pituitary adenylate cyclase activating polypeptide can regulate testicular germ cell protein synthesis in vitro.

The aim of this study was to explore whether pituitary adenylate cyclase activating polypeptide (PACAP) could regulate protein synthesis by enriched preparations of spermatocytes and spermatids from the adult rat testis. Spermatocytes and spermatids were incubated for 8 h or 24 h in the absence (control) or presence of PACAP-27, PACAP-38, vasoactive intestinal peptide (VIP) or dibutyryl adenosine-3',5'-cyclic monophosphate (db-cAMP). Total synthesis of intracellular and secreted proteins, during the incubation periods, was assessed and selected samples were analysed by 2-D SDS-PAGE. PACAP-38 (200 nM), VIP (200 nM) and db-cAMP (1 mM) significantly increased the synthesis of spermatocyte-secreted and intracellular proteins by 8 h and 24 h. Synthesis of both intracellular and secreted proteins by spermatids was significantly inhibited at 8 h and 24 h with PACAP, VIP and db-cAMP. The abundance of four germ cell-secreted proteins (GSP1, GSP2, GSP3 and phosphatidyethanolamine-binding protein (PEBP), which can be identified in both spermatocyte and spermatid culture medium, and beta-actin, which can only be identified in spermatid culture medium, was analysed. PACAP-38 and db-cAMP significantly increased the incorporation of label into GSP1, GSP2, GSP3 and PEBP, derived from spermatocyte culture medium, at 8 h and 24 h. In contrast PACAP-38 inhibited the incorporation of label into GSP1 and beta-actin, derived from spermatid culture medium, at 24 h. The results show that PACAP can regulate synthesis of both secreted and intracellular proteins by spermatids and spermatocytes in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Differential regulation of cyclic adenosine 3',5'-monophosphate (cAMP) response element-binding protein and cAMP response element modulator messenger ribonucleic acid transcripts by follicle-stimulating hormone and androgen in the adult rat testis.

Hormonal regulation of the expression of mRNA transcripts for cAMP response element-binding protein (CREB) and cAMP response element modulator (CREM) during spermatogenesis was studied in the adult rat testis. Northern analysis of CREB and CREM identified two mRNA transcripts for CREM (2.4 and 1.6 kb) and one transcript for CREB (2.0 kb). Analysis of mRNAs from isolated testicular cells by reverse transcriptase polymerase chain reaction (RT/PCR) showed that CREM mRNAs were expressed by the germ cells but not the Sertoli or interstitial cells, whereas CREB mRNA was located in germ cells, Sertoli cells, and interstitial cells. RNA was isolated and analyzed from the testes of 1) rats treated for 24 h with FSH, 2) rats in which androgen withdrawal had been induced by ethane dimethane sulphonate (EDS) treatment 6 days earlier (EDS-treated), 3) EDS-treated rats supplemented with testosterone (EDS + T), or 4) intratesticular administration or dibutyryl cAMP (dbcAMP) in the preceding 24 h. CREM mRNA transcript expression was found to be decreased after all of these treatments in samples from intact testis and from isolated cells. Expression of the CREB transcript was also decreased by EDS-induced androgen withdrawal, but not by FSH or EDS + T. In situ hybridization of paraffin-embedded testis sections probed with digoxigenin-labeled riboprobes confirmed the localization of CREB and CREM mRNA to the same cell types as found with RT/PCR. No stage-dependent expression of CREM mRNA transcripts could be observed. Hybridization of the CREB probe was highest around the base of stage VII-VIII tubules, and this was shown to be androgen-dependent. The data presented suggest that regulation of the expression of CRE-binding protein mRNAs in Sertoli and germ cells during spermatogenesis is dependent on both androgen and FSH. However, the effects of androgen or FSH on the regulation of CRE-binding protein mRNAs are different.

Androgens↗
PubMed1994

Localization of mRNAs by in-situ hybridization to the residual body at stages IX-X of the cycle of the rat seminiferous epithelium: fact or artefact?

Several recent articles have reported localization of specific mRNAs in the rat testis to stage IX and X seminiferous tubules using in-situ hybridization. In all cases the expression was located basally in the tubules and appeared as discrete round clusters of grains close to the lamina propria. The localization was interpreted as being in Sertoli cells or leptotene spermatocytes. In this study we demonstrate that this pattern is most probably due to artefactual binding of probes to the residual body (RB). In the present study testicular tissue, perfusion-fixed with Bouin's and embedded in paraffin, was used, as this resulted in excellent morphological preservation such that RBs within tubules at stages VIII-X were clearly distinguishable. RNA content of the RBs was demonstrated at stages VIII-X using methyl green pyronin staining, and could be eliminated by pretreatment with RNAse or trichloroacetic acid. Localization of mRNAs for 11 seminiferous tubule proteins was assessed using 35S-labelled and digoxigenin-labelled riboprobes (activin receptor-II, alpha-inhibin, transferrin, androgen-binding protein (ABP), cyclic protein-2 (CP-2), CREM, sulphated glycoproteins 1 and 2 (SGP-1 and SGP-2), transition protein 2 (TP-2) and cystatin-C), and digoxigenin-labelled oligonucleotide probes (transition protein-1 (TP-1), TP-2 and protamine-1). All of these probes showed localization to the correct cell type(s) within the seminiferous epithelium. In addition, six antisense riboprobes (activin receptor-II, CREM, SGP-2, CP-2, cystatin C and alpha-inhibin) showed hybridization to basally located residual bodies in tubules at stages IX-X on one or more occasions, whereas residual bodies around the edge of the lumen (stage VIII) or in transit through the seminiferous epithelium showed no hybridization; sense probes showed no localization to residual bodies. A common feature of the probes which localized to the basal RBs was that they had been prepared using cDNA cloned into Bluescript SK- vector such that the antisense strand was generated from the T7 polymerase promotor. A cRNA prepared using T7 polymerase and Bluescript vector alone and a GC-rich 27mer oligonucleotide corresponding to the region of the multiple cloning site of Bluescript adjacent to the T7 site both localized uniquely to basal RB. It is concluded that the hybridization seen within RBs is probably a subtle artefact unique to RBs undergoing dissolution following fusion with Sertoli cell lysosomes, and may reflect nonspecific hybridization to GC-rich fragments of RNA.

Animals↗