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Biomedical subjects

R M Sharpe

Publications and source records attributed to R M Sharpe.

At least 73 records · Page 4Linked to original sources
PubMed1994

Immunohistochemical localization of androgen receptors in the rat testis: evidence for stage-dependent expression and regulation by androgens.

Androgens are essential for the maintenance of normal spermatogenesis in the rat. We assessed the sites, developmental pattern, and hormonal control of androgen receptors (AR) in the rat testis. Adult male rats were studied after 1) no treatment; 2) ethane dimethane sulfonate (EDS), which eradicates Leydig cells and endogenous testosterone (T); 3) EDS plus T replacement beginning at the time of EDS administration; or 4) methoxyacetic acid, which leads to the loss of specific germ cell types. Testes were also obtained from normal immature rats (aged 5, 14, 16, 21, 28, 31, 35, 38, and 45 days). After microwave antigen retrieval, immunohistochemistry was performed using a rabbit polyclonal antibody (Novocastra) raised against a peptide unique to the N-terminal region of the AR and detection with biotinylated swine antirabbit immunoglobulin G, avidin-biotin complex/alkaline phosphatase, and nitroblue tetrazolium salt (NBT)/5 bromo-4-chloro-3-indolylphosphate (BCIP) substrate. In adults, nuclear immunostaining of Sertoli cells (SC) increased progressively in intensity from stages II through VII of the spermatogenic cycle, and then declined precipitously during stage VIII to become barely detectable in stages IX-XIII. Prominent AR immunostaining was also evident in peritubular myoid cells, arterioles, and interstitial cells; staining in these cells did not vary with the stage of the cycle of the adjacent tubules. EDS caused a severe loss of AR immunostaining in all cell types. Replacement of T in EDS-treated animals resulted in a pattern of AR immunostaining comparable to that in controls, although staining intensity was reduced. Methoxyacetic acid administration did not affect the pattern of AR staining. In immature rats, peritubular myoid cell immunostaining was prominent from day 5; SC staining was detectable on day 5, increased in intensity with age, and became stage dependent between days 21-35. The following conclusions were reached. 1) Immunohistochemically detectable AR expression in SC occurs predominantly in stages II-VII of the spermatogenic cycle, with highest levels at stage VII. 2) AR immunostaining is also prominent in peritubular myoid cells, arterioles, and Leydig cells (but not in germ cells), but is unrelated to the stage of adjacent tubules. 3) Endogenous T and/or its metabolites control the expression of AR in the testis. 4) AR immunostaining is detectable by day 5 of age and becomes stage specific in SC between days 21-35.

Acetates↗
PubMed1994

Temporal relationship between androgen-dependent changes in the volume of seminiferous tubule fluid, lumen size and seminiferous tubule protein secretion in rats.

This study assessed whether changes in production of seminiferous tubule fluid underlie the previously described androgen-dependent changes in protein secretion by seminiferous tubules at stages VI-VIII of the spermatogenic cycle. Testosterone withdrawal was induced in adult rats by administration of ethane dimethane sulfonate (EDS) and temporal changes in lumen area, the volume of seminiferous tubule fluid and testicular interstitial fluid were assessed and compared with the changes in secretion of [35S]methionine-labelled proteins in vitro by isolated seminiferous tubules at stages VI-VIII. Testicular interstitial fluid was reduced by about 50% by day 4 and later after EDS treatment when compared with controls. In contrast, the volume of seminiferous tubule fluid was unaffected at days 3 and 4 but was reduced by about 50% at days 6 and 8 after EDS treatment. In perfusion-fixed control testes, the lumen area of seminiferous tubules at stages VII-VIII was significantly greater than at stages I-VI and IX-XIV. This difference was also evident at 4 days after EDS treatment, but was abolished at 6 and 8 days after treatment. The volume of testicular interstitial fluid was reduced significantly at 3 and 4 days after EDS treatment, but was further reduced (about 50%) at 6 and 8 days. Administration of 25 mg testosterone esters every 3 days to EDS-treated rats prevented all of the changes described above.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Identification of stage-specific changes in protein secretion by isolated seminiferous tubules from rats following exposure to short-term local testicular heating.

The objective of this study was to identify the early (after 4 and 24 h) effects of short-term local testicular heating (43 degrees C for 30 min) on the secretion of proteins by seminiferous tubules isolated from adult rats at stages II-V, VI-VIII or IX-XII of the spermatogenic cycle, and cultured in vitro for 24 h with [35S]methionine. Incorporation of [35S]methionine into secreted and intracellular proteins was assessed and the pattern of protein secretion was evaluated using two-dimensional SDS-PAGE. Seminiferous tubules isolated from control rats exhibited the characteristic, androgen-dependent increase in protein secretion at stages VI-VIII. At 4 h after exposure to local testicular heating, seminiferous tubules at these stages showed a significant increase (P < 0.001) in the overall incorporation of [35S]methionine into secreted proteins, whereas seminiferous tubules at stages II-V and IX-XII showed no significant change. In marked contrast, seminiferous tubules isolated from rats 24 h after local testicular heating showed a significant decrease in the incorporation of [35S]methionine into secreted proteins at stages VI-VIII (P < 0.001) and to a lesser extent at IX-XII (P < 0.05), whereas seminiferous tubules at stages II-V showed no change in incorporation. Prior treatment to maintain normal intratesticular concentrations of testosterone in heat-exposed rats failed to prevent these changes. Similar results were obtained when incorporation of [35S]methionine into intracellular proteins was evaluated 4 and 24 h after exposure to local testicular heating.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Are oestrogens involved in falling sperm counts and disorders of the male reproductive tract?

The incidence of disorders of development of the male reproductive tract has more than doubled in the past 30-50 years while sperm counts have declined by about half. Similar abnormalities occur in the sons of women exposed to diethylstilbestrol (DES) during pregnancy and can be induced in animals by brief exposure to exogenous oestrogen/DES during pregnancy. We argue that the increasing incidence of reproductive abnormalities in the human male may be related to increased oestrogen exposure in utero, and identify mechanisms by which this exposure could occur.

Animals↗
PubMed1993

Stage-dependent changes in spermatogenesis and Sertoli cells in relation to the onset of spermatogenic failure following withdrawal of testosterone.

Rapid and complete withdrawal of intratesticular testosterone was achieved via the destruction of all Leydig cells with the specific Leydig cell cytotoxin ethane dimethanesulphonate (EDS). Restoration of testosterone levels was accomplished by administration of a single dose (25 mg) of testosterone esters (T) known to reverse the antispermatogenic effects of androgen withdrawal. Quantitation of the degenerating germ cells in cross sections of seminiferous tubules (ST) at stages IV-V, VII, IX, and X-XI of the spermatogenic cycle was used as a sensitive biological index of the effects of testosterone withdrawal and restoration upon the function of the Sertoli cells. Compared to control testicular tissues, the mean numbers of pyknotic germ cells per ST cross section at stages VII, IX and X-XI increased significantly (P < 0.01-0.001) between 4 to 8 days post-EDS treatment, but only in stage VII tubules was this trend reversed significantly (P < 0.005) within 2 days by T supplementation. In EDS-treated rats, stages VII, VIII, IX, and X-XI also exhibited significant (P < 0.05-0.001) increases (compared to controls) in the volumetric proportions by which intraepithelial vacuoles appeared within the seminiferous tubules. Again, in EDS+T supplemented rats, the appearance of vacuoles was significantly (P < 0.001) suppressed in stage VII and VIII. In contrast to tubules at stages VII-XI, those at stages IV-V were completely unaffected by testosterone withdrawal or replacement. The results show that at selected time intervals after EDS treatment, testosterone supplementation is capable of preventing/reversing these morphological changes within 2 days in stage VII tubules. It is suggested that the induction and subsequent prevention of seminiferous epithelial damage will serve as an important in vivo and in vitro approach for studies on the androgen-mediated changes in Sertoli cell biology during phases of impairment and recovery of their function. Manipulation of adult Sertoli cell function as provided by our model should permit identification of androgen-regulated gene products together with an understanding of their role(s) in normal and abnormal spermatogenesis.

Animals↗
PubMed1993

Identification of stage-specific changes in protein secretion by isolated seminiferous tubules from the rat following exposure to either m-dinitrobenzene or nitrobenzene.

The objective of this study was to identify effects of two known Sertoli cell toxicants on the secretion of proteins by seminiferous tubules (ST) isolated from adult rats at different stages of the spermatogenic cycle and cultured in vitro for 24 hr with [35S]methionine. Adult rats received a single oral dose of 50 mg/kg metadinitrobenzene (m-DNB) or 300 mg/kg nitrobenzene (NB). Long lengths of ST at stages II-V, VI-VIII or IX-XII were then isolated from control and treated rats at 1 or 3 days post-treatment; selection of stages was based on the stage specificity of the early (24-72 hr) adverse effects of m-DNB and NB on spermatogenesis in vivo. In addition, ST at the same stages were isolated from untreated rats and cultured in the presence or absence of m-DNB or NB (10(-4)M). Incorporation of [35S]methionine into secreted proteins was assessed and the pattern of protein secretion evaluated using two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2-D SDS-PAGE). ST isolated from rats pretreated 24 hr earlier with NB in vivo showed a significant decrease in the overall incorporation of [35S]methionine into secreted proteins at stages VI-VIII and IX-XII, whereas ST at stages II-V showed no such change; comparable protein changes were observed when 10(-4) M NB was added in vitro for 24 hr to ST isolated at the same stages from untreated rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Cellular localisation of messenger RNAs in rat testis: application of digoxigenin-labelled ribonucleotide probes to embedded tissue.

In an attempt to identify key changes involved in normal spermatogenesis we have developed methods to enable the study of gene expression by the various subpopulations of testicular cells by use of in-situ hybridisation histochemistry. The use of digoxigenin-labelled ribonucleotide and oligonucleotide probes on testicular tissue perfusion-fixed with Bouin's fixative and embedded in paraffin, polystyrene or methacrylate, has been used to accurately localise three transcripts to three different cell types (Sertoli cells, pachytene spermatocytes, and step 7-12 spermatids) within the seminiferous tubule. The ability to produce semi-thin sections of polystyrene- or methacrylate-embedded tissue and successfully to apply digoxigenin-labelled ribonucleotide or oligonucleotide probes resulted in far greater resolution and unequivocal localisation of mRNA in testicular cells than was previously possible by use of thicker paraffin or frozen sections hybridised with 35S-labelled riboprobes. A comparison of the different embedding media versus digoxigenin-labelled oligonucleotide or ribonucleotide probes is made and we demonstrate the relative sensitivities and merits of each combination.

Animals↗
PubMed1993

Response of the seminiferous epithelium of the rat testis to withdrawal of androgen: evidence for direct effect upon intercellular spaces associated with Sertoli cell junctional complexes.

The morphological response of the Sertoli cells to partial or complete withdrawal of testosterone was studied in adult rats following hypophysectomy or administration of ethane dimethanesulphonate (EDS), a toxicant known to destroy selectively the Leydig cells of the testis. To assess the role of germ cells in effecting changes to Sertoli cells following withdrawal of testosterone, germ cell-deficient rats with Sertoli-cell-only testes (SCO) were treated with EDS to remove the source of testosterone. At 6 days after hypophysectomy or 4, 6 and 8 days after EDS treatment, stage VII and VIII seminiferous tubules showed degenerating germ cells and numerous basally-located vacuoles approximately 1-15 microns in diameter. Ultrastructural analysis indicated that most of the vacuoles were multiple focal dilations of the intercellular space associated with Sertoli cell junctional complexes. In SCO rats, treatment with EDS resulted in a significant (P < 0.05) increase in the formation of many vacuoles particularly in the base but also in the trunk of the Sertoli cells and again electron microscopic analysis showed multiple, localized expansions of the intercellular space associated with Sertoli cell junctional complexes. The appearance of intercellular spaces in SCO testes following androgen withdrawal cannot be attributed to shrinkage of degenerating germ cells since the seminiferous tubules did not contain germ cells. It is concluded that withdrawal of androgen induces early morphological alterations of the Sertoli cell junctional complexes in which the sites of membrane fusions representing tight junctions remain intact whereas the intercellular spaces exhibit major focal dilations.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Stage-dependent expression of mRNA for cyclic protein 2 during spermatogenesis is modulated by elongate spermatids.

Cyclic protein 2 (CP-2) is a product of the Sertoli cell which is secreted in a cyclical manner according to the stage of the spermatogenic cycle. This study has assessed the influence of the germ cell complement on expression of CP-2 mRNA. Adult rats were treated with 650 mg/kg methoxyacetic acid (MAA) to induce the specific depletion of > 80% of pachytene and later spermatocytes from most tubules, and expression of CP-2 mRNA was then assessed at various times after treatment when particular germ cell types were depleted selectively. CP-2 mRNA was specifically localised to the Sertoli cells of the seminiferous tubules by non-radioactive in situ hybridisation using a digoxigenin-labelled riboprobe. A stage specific variation in CP-2 mRNA levels was observed, with the mRNA being most abundant at stages IV-VII of the spermatogenic cycle. Northern analysis revealed that treatment with MAA led to an apparent increase in the amount of the major 1.7 kb CP-2 transcript when either pachytene spermatocytes or round spermatids were depleted. In contrast, the level of CP-2 mRNA was decreased by more than half at 21 days after MAA treatment. This decrease was confirmed by in situ hybridisation at 21 days after MAA treatment, when CP-2 mRNA expression was found to be decreased or absent from tubules at stages at which CP-2 mRNA is normally expressed (stages IV-VII) when elongate spermatids were depleted selectively from these tubules. These observations lead us to hypothesise that elongate spermatids positively modulate CP-2 mRNA expression in the Sertoli cell.

Acetates↗
PubMed1993

Mitochondrial cytochrome C oxidase II messenger ribonucleic acid is expressed in pachytene spermatocytes at high levels and in a stage-dependent manner during spermatogenesis in the rat.

Screening of a rat seminiferous tubule library (stages VI-VIII) resulted in isolation of several clones to the same abundant mRNA. Sequencing revealed that these encoded all or part of the second subunit of cytochrome C oxidase (COX II), the terminal enzyme in the electron transport chain located in mitochondria. The pattern of expression of the COX II mRNA in the testis was examined by Northern blot and in situ hybridisation combined with specific depletion of pachytene spermatocytes or Leydig cells by treatment with methoxyacetic acid (MAA) or ethane dimethane sulphonate (EDS), respectively. COX II mRNA was found to be strongly expressed in pachytene spermatocytes in adult and prepubertal rats, with the highest levels of expression at stages VII-VIII of the spermatogenic cycle. Although this is the androgen-dependent stage, Northern analysis of total testicular RNA showed that expression of COX II mRNA was not altered detectably by EDS-induced androgen withdrawal nor was expression altered detectably by 24-h treatment with high doses of FSH. Treatment of rats with MAA, which selectively depletes seminiferous tubules at most stages of pachytene spermatocytes, resulted in a marked reduction in COX II mRNA. In the absence of pachytene spermatocytes, COX II mRNA was visualized in preleptotene spermatocytes and in Sertoli cell cytoplasm. The expression of COX II in Sertoli cells was confirmed by examination in situ of a testis devoid of most germ cells and by Northern analysis of RNA from an adult Sertoli cell-enriched preparation. However, using either approach the amount of mRNA in Sertoli cells was considerably lower than that in pachytene spermatocytes. Cytochrome C oxidase has thirteen subunits, three of which (I-III) are encoded on the mitochondrial genome. The physiological significance of the high levels of expression of COX II mRNA in pachytene spermatocytes and its stage-dependent changes is unknown but they presumably reflect requirements for alterations in energy demand as these cells enter the final stages of meiosis. The stage-dependent differences in expression of COX II mRNA in pachytene spermatocytes may also explain differences in the susceptibility of these cells to depletion by MAA.

Animals↗
PubMed1993

Relative roles of testosterone and the germ cell complement in determining stage-dependent changes in protein secretion by isolated rat seminiferous tubules.

This study has compared the effect of withdrawal of testosterone (+/- replacement) with that of selective depletion of pachytene spermatocytes (PS) or round/elongating spermatids (RS), or both PS and RS, on the level of overall protein secretion by seminiferous tubules (ST) isolated at particular stage-groups of the spermatogenic cycle. Testosterone withdrawal was induced by destroying the Leydig cells with a single injection of ethane-dimethane sulphonate (EDS), with or without concomitant replacement of testosterone by injection; ST at stages II-V, VI-VIII or IX-XII were then isolated from control and treated rats at 4 days after treatment. Methoxyacetic acid (MAA) was administered, in either one or two doses, to selectively destroy 80-100% of pachytene and later spermatocytes; ST at stages I-V, VI-VIII or IX-XIV were then isolated at specific times after treatment such that ST were depleted selectively of either PS, RS or PS+RS. Isolated ST (5 cm) were then cultured for 22 h at 34 degrees C with 35S-methionine and its incorporation into secreted proteins then quantified. Based on the incorporation of 35S-methionine, ST at stages VI-VIII showed a significantly higher level of protein secretion than did ST at earlier or later stages. This difference was abolished following testosterone withdrawal but was maintained by testosterone replacement. The normal increase in protein secretion by ST at stages VI-VIII was also prevented if either PS or RS were depleted, whereas depletion of either PS or RS alone had no significant effect on protein secretion by ST at stages I-V, and only the depletion of RS significantly reduced protein secretion by ST at stages IX-XIV. Depletion of both PS+RS reduced protein secretion significantly by ST at all stages. In contrast to the data for total protein secretion, the levels of sulphated glycoprotein (SGP)-1 and SGP-2 secreted by ST at stages VI-VIII showed that these two Sertoli cell proteins were unaffected by germ cell depletion except after co-depletion of PS+RS when secretion of SGP-1 was halved.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetates↗
PubMed1993

Intratesticular hormone levels and the route of secretion of hormones from the testis of the rat, guinea pig, monkey and human.

Blood samples were obtained from the testes of rats, guinea pigs and Macaque monkeys and from normal men undergoing vasectomy reversal, in order to assess the comparative dynamics of hormone secretion. In each species, blood was sampled from a vein on the surface of the testis (testicular venous blood, TV), from a vein in the spermatic cord above the pampiniform plexus (spermatic venous blood, SV) and from a vein elsewhere in the body (peripheral venous blood, PV). Plasma concentrations of testosterone and inhibin were then determined by radioimmunoassay. In all species, testosterone secretion profiles were comparable, with concentrations being greatest in TV blood. SV concentrations were reduced by 40-60% compared with TV levels, with a significantly greater reduction in PV levels. Inhibin secretion varied significantly between species, with the rat being the only animal to show significant increases in inhibin concentrations from TV to SV blood. Inhibin secretion in the guinea pig was most comparable with that of the rat, although the increased SV levels fell short of being significant. Macaque and human profiles contrasted with those of the rat and guinea pig, with the greatest inhibin concentrations being found in TV blood. Levels in SV blood were reduced by some 40%, and PV levels were reduced significantly further. These differences may be due to the different position of the rete testis, and its relationship to the testicular vasculature, in these species. The sampling procedure described provides a defined set of testicular blood samples that could contribute important information of relevance to physiological and clinical studies of testicular function.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1993

Evaluation of possible determinants and consequences of Leydig cell heterogeneity in man.

Leydig cells in the human testis are highly heterogeneous, consisting of variably staining light and dark cells. The basis for this difference is unknown. The present study has assessed whether differing numbers or proportions of dark and light Leydig cells are related: (1) to the pronounced inter-individual variation in testosterone production by isolated Leydig cells, and (2) to differences in structural composition of the testis. Testes (paired weight 6.6-59.48 g) were obtained from 27 men aged 72.9 +/- 9.5 years (range 54-89 years) undergoing orchidectomy as primary treatment for prostatic cancer. Leydig cells were isolated by Percoll-purification and cultured for 20 h under basal and hCG-stimulated conditions. The proportion of light and dark Leydig cells isolated by this method was shown to reflect their proportions in situ, based on the morphometric analysis of fixed testicular tissue from the same men. Leydig cells isolated from all testes produced testosterone in vitro and responded to stimulation by hCG, though the amounts of testosterone produced varied widely between subjects. Because of the latter, samples were grouped into 'low' (n = 9), 'medium' (n = 11) and 'high' (n = 7) groups on the basis of their testosterone production. These groups did not differ in their age, testicular size or gross testicular morphology, though men in the 'high' group tended to have more total Leydig cells per testis. However, there was no overall correlation between testosterone production by isolated Leydig cells and the numbers of light or dark Leydig cells or their ratio or the total number of Leydig cells per testis. The relationship between the volume of light and dark Leydig cells and testicular composition was also assessed. The volume of both types of Leydig cells was strongly correlated (p < 0.001) with the volume of germ cells, but otherwise light and dark Leydig cells correlated positively with different structures. Thus, the volume of light Leydig cells correlated (p < 0.001) with the volume of blood vessels and of peritubular tissue whereas the volume of dark Leydig cells correlated (p < 0.01) with that of the tubular lumen. These differences could indicate differences in regulation and/or function of light and dark Leydig cells. However, the present data do not support the idea that light and dark Leydig cells may differ in their steroidogenic capacity.

Aged↗
PubMed1993

Effect of age on seminiferous tubule protein secretion and the adverse effects of testicular toxicants in the rat.

This study has assessed the effect of age on protein secretion by seminiferous tubules (ST) isolated from rats and their response to Sertoli cell toxicants. ST were isolated from immature (aged 28 days), late pubertal (aged 45 days) and young adult (aged 70 days) rats and cultured in vitro for 24 h with 35S-methionine in the presence or absence of FSH (1 mg/ml), m-dinitrobenzene (m-DNB) or nitrobenzene (NB) (both at 10(-4)M). Incorporation of 35S-methionine into newly synthesized proteins in the culture medium (secreted proteins) was assessed and the pattern of protein secretion evaluated using two-dimensional sodium dodecylsulphate polyacrylamide gel electrophoresis (2-D SDS-PAGE). These data were compared with those obtained using cultures of immature rat Sertoli cells+germ cells (SC+GC). Addition of FSH, m-DNB or NB in vitro either had no effect (NB) or had a small stimulatory effect (FSH and m-DNB) on the incorporation of 35S-methionine into overall secreted proteins by ST isolated from immature rats. At the same doses, addition of either FSH, m-DNB or NB to SC+GC co-cultures resulted in increased incorporation of radiolabel into secreted proteins in all instances. In contrast, the same additions to ST isolated from adult rats resulted in a 20-34% decrease in the overall incorporation of 35S-methionine into secreted proteins. ST isolated from late pubertal rats showed a similar response to adult rats except that the decreases in incorporation induced by FSH, m-DNB and NB were smaller. Analysis by 2-D SDS-PAGE revealed considerable age-dependent differences in the proteins secreted by ST from immature and adult rats, of which 13 were identified as being of potential importance. Most of these proteins were prominent secretory products of ST from adult rats, but were minor or non-detectable products of cultures of ST or SC+GC from immature rats. Most of these proteins disappeared or decreased in abundance after culture of ST with m-DNB or NB. Two proteins showed the reverse pattern, being more prominent secretory products in immature than mature rats, and their secretion was unaffected or was increased by toxicant exposure. These results demonstrate that there are major age-dependent differences in the secretion of total and specific proteins by isolated ST and that these are probably related to changes in the germ cell complement with age.(ABSTRACT TRUNCATED AT 400 WORDS)

Aging↗
PubMed1992

Stage-specific expression of rat transition protein 2 mRNA and possible localization to the chromatoid body of step 7 spermatids by in situ hybridization using a nonradioactive riboprobe.

The present study has used methoxyacetic acid (MAA)-induced depletion of specific germ cell types in the rat and in situ hybridization with nonradioactive riboprobes to determine the stages of the spermatogenic cycle at which there is expression of the mRNA for the basic chromosomal protein transition protein 2 (TP2). On Northern blots, an abundant mRNA was detectable in samples from control adult rats, but the amount of message was markedly reduced when RNA was extracted from the testes of rats treated 14 and 21 days previously with methoxyacetic acid. These testes were depleted specifically of step 7-12 spermatids, suggesting that these cells contain TP2 mRNA. When tissue sections were subjected to in situ hybridization, the TP2 mRNA was localized at the cellular and subcellular levels. Messenger RNA for TP2 was first detectable in spermatids at step 7. In these spermatids, at high magnification, in addition to some positive reaction in the cytoplasm, intense staining was located to a perinuclear structure consistent with localization of mRNA within the chromatoid body. The amount of TP2 mRNA in the cytoplasm increased as remodelling of the early spermatid nucleus progressed and was highest in step 10 and 11 spermatids at stages X and XI. Thereafter, the mRNA decreased until it was undetectable in step 14 spermatids at stage XIV. The localization of TP2 mRNA to the chromatoid body of step 7 spermatids would be consistent with this organelle being a storage site for long-lived mRNAs utilized later in spermiogenesis.

Animals↗
PubMed1992

Testosterone and FSH have independent, synergistic and stage-dependent effects upon spermatogenesis in the rat testis.

Adult rats were hypophysectomized and treated with ethane dimethanesulphonate (EDS) selectively to eliminate the Leydig cells in the testis. By removing the source of endogenous gonadotrophins and androgens, the subsequent effects on the seminiferous epithelium were studied after 20 days of treatment with vehicle, or FSH (2 x 50 micrograms/day) or a low dose of testosterone (0.6 mg testosterone esters every 3rd day) alone or in combination. Compared to vehicle-treated hypophysectomized rats with Leydig cells, testis weight in saline-treated hypophysectomized rats treated with EDS declined by 50%, spermatogenesis was disrupted severely and only 18% of the tubules contained spermatids, these being confined to stages I-VI of the spermatogenic cycle. Treatment with either FSH or testosterone esters alone significantly (P less than 0.01) increased testis weight compared to vehicle-treated hypophysectomized rats treated with EDS and 40% of tubules contained spermatids either at stages I-VI after FSH, or at all stages I-XIV after testosterone treatment. Treatment with FSH and testosterone esters together maintained testis weights approximately 20% above vehicle-treated hypophysectomized controls; over 70% of the seminiferous tubules contained spermatids and there was a marked stimulation of spermatogenesis at all stages of the spermatogenic cycle. The results suggest, that in the absence of the pituitary gland and the Leydig cells, FSH alone partially supports spermatogenesis up to the development of round spermatids whereas testosterone is capable of maintaining spermatid development at all 14 stages of the cycle. When FSH and testosterone were administered in combination, the effects upon spermatogenesis were far greater than the response expected if their individual effects were simply additive. It is therefore concluded that FSH may play a role in normal spermatogenesis and that this role is essentially that of augmenting the response of the testis to testosterone. The biochemical mechanisms via which this might occur are discussed and hypophysectomized rats treated with EDS used in the present studies should provide a useful approach for their identification.

Animals↗
PubMed1992

The role of specific germ cell types in modulation of the secretion of androgen-regulated proteins (ARPs) by stage VI-VIII seminiferous tubules from the adult rat.

This study has assessed the role of specific germ cell types in modulating the secretion of seven previously identified androgen-regulated proteins (ARPs) as well as the increase in overall level of protein secretion by seminiferous tubules (ST) at stages VI-VIII of the spermatogenic cycle in the rat. Methoxyacetic acid (MAA) was administered at 650 mg/kg to induce 80-100% depletion of pachytene and later spermatocytes; ST at stages VI-VIII were then isolated at 4, 18 and 30 days after MAA treatment when pachytene spermatocytes (PS), round spermatids (RS) or elongate spermatids (ES), respectively, were depleted selectively. By comparison with ST isolated from controls or from rats 4 days after ethanedimethane sulphonate (EDS)-induced testosterone withdrawal, the overall level of incorporation of 35S-methionine into ST-secreted proteins was assessed after culture for 22 h at 32 degrees C as well as the relative abundance of ARPs identified by two-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Depletion of PS caused a substantial decrease in abundance of ARP-6 (53-57 kDa; pI 5.7-5.8) and ARP-7 (56-59 kDa; pI 5.9-6.2) and minor and more variable decreases in ARP-3 (27-30 kDa; pI 7.4) and ARP-4 (38 kDa; pI 6.0-6.2); ARP-6 and ARP-7 are comparable in size to the A and B forms of P-Mod-S, a product of the peritubular cells. Depletion of RS resulted in the complete disappearance of ARP-2 (13-14.5 kDa; pI 7.3-7.6), a decrease in abundance of ARP-7 and variable decreases in ARP-1 (approximately 9.5 kDa; pI 6.9-7.4) and ARP-6; ARP-1 and -2 are thought to be Sertoli cell-secreted proteins. Depletion of ES had no effect on secretion of the ARPs. Depletion of PS, RS or ES resulted in significant (P less than 0.001) decreases of 26, 38 and 42% respectively in the overall level of incorporation of methionine into ST-secreted proteins, but this was significantly less than the decrease (55%) induced by EDS-induced testosterone withdrawal. These results show that the androgen-dependent changes in secretion of proteins, which occur specifically at stages VI-VIII of the normal spermatogenic cycle, are largely, if not completely, dependent on a normal germ cell complement, with each germ cell type regulating the secretion of proteins essential for its own development.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetates↗