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H. P. Bugge and L. Plöen

Summary. Volume density of Sertoli cells in mature rats was estimated in different stages of the cycle of the seminiferous epithelium. Manual point-counting on electron micrograph montages revealed a more than 40% increase in Sertoli cell volume from just before to just after spermiation. It is suggested that this variation should be considered in studies of stage-dependent cyclic variations in activity of Sertoli cells and/or germ cells.

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J. L. Courtens, L. Plöen and M. Loir

Summary. Protamine was specifically demonstrated in spermatids and spermatozoa of the boar by immunoelectron microscopy, using anti-boar or anti-ram protamine antisera, and three different direct or indirect labelling techniques. The two isomers of the protamine could not be labelled separately. The protamine is present in the cytoplasm of elongating spermatids and it enters the nuclei throughout the elongation process after possible storage in the cytoplasm or in the nuclear envelope of spermatids, or both. These findings differ from previous observations in other species.

Keywords: spermiogenesis; spermatozoa; protamine; immunocytochemistry; electron microscopy; boar

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BP Setchell, L Ploen and EM Ritzen

The effects of local heating of rat testes, in which spermatogenesis had been suppressed with injections of a GnRH agonist and an anti-androgen, were examined. Although the detrimental effects of heating were not as marked as those found in the testes of non-injected rats, the testes in which spermatogenesis was suppressed also showed a significant reduction in mass, the number of spermatozoa, tubular diameter and the percentage of normal tubular cross-sections at day 35 after heating. The results indicate that heating has an effect on cells in the testis other than those shown to be most susceptible to heat, namely pachytene spermatocytes and early spermatids, which were absent or markedly reduced in number when spermatogenesis was suppressed. The long-term effects of heating on the above parameters, as reported in a previous study, were also confirmed. However, in testes in which spermatogenesis was suppressed at the time of heating, there appeared to be no or a reduced long-term impairment of spermatogenesis, as determined by testis mass, the percentage of qualitatively normal tubules and epididymal sperm counts.

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BP Setchell, L Ploen and EM Ritzen

Heating the testes of anaesthetized adult rats to 43 degrees C for 30 min in a waterbath was followed by a large decrease in testis and epididymis mass and number of spermatozoa 35 days later. These parameters had recovered to some extent, but not completely, by days 70 and 97 after heating, but had decreased again in rats examined on day 182. There were no consistent effects of heating on androgen status, as determined by the concentrations of testosterone in blood and testis fluids, or by seminal vesicle mass, and interstitial fluid volume was increased in the heated testes. Treatment of rats with an implant of a GnRH agonist and daily injections of an anti-androgen for 14 days (sufficient in itself to cause large temporary decreases in tissue mass, number of spermatozoa and androgen status) did not reduce the initial decrease in testis mass or number of spermatozoa seen after heating, but reduced the later decreases in mass and number of spermatozoa significantly. These findings indicate that, as well as causing damage to spermatocytes and spermatids, as previously reported, heating also reduces the ability of spermatogonia to repopulate the seminiferous tubules at longer intervals after heating. Furthermore, it appears that this effect on the spermatogonia can be reduced by treating the animals with a GnRH agonist and anti-androgen, a treatment similar to that shown by other authors to improve recovery of the testis from irradiation or drug treatment.

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H. Rodriguez-Martinez, J. L. Courtens, U. Kvist and L. Plöen

Summary. Protamine was specifically demonstrated in boar spermatozoa collected from the rete testis, caput, corpus and cauda epididymidis and the ejaculate by immunoelectron microscopy, using anti-boar or anti-ram protamine antisera and an indirect post-embedding immunogold technique. Spermatozoa from all collection sites stained after incubation although with different degrees of labelling. Controls were negative. Labelling increased from the rete testis towards the epididymal corpus, where it was most intense, decreasing sharply thereafter. The weakest binding of the assayed antibodies was obtained in the ejaculated spermatozoa but it could be reversed by in-vitro induction of chromatin decondensation with sodium dodecyl sulphate and the metal-chelating EDTA. The finding of a significant decrease in the immunolabelling detected from the corpus epididymidis onwards indicates a critical point for the interaction between DNA and the protamines in boar spermatozoa during the epididymal maturation.

Keywords: spermatozoa; epididymis; protamine; immunocytochemistry; electron microscopy; boar

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J. L. Courtens, H. Ekwall, M. Paquignon and L. Plöen

Summary. Boar semen was analysed by electron microscopy coupled to image analysis and X-ray energy dispersive spectroscopy, during the usual process for freezing and thawing in field conditions. Freeze–substitution and freeze–quenching permitted recording of real or potential intracellular ice before, during, and after freezing. Heads and flagella displayed two different osmotic properties before freezing. Heads were dehydrated progressively before and during freezing, while flagella were hydrated before freezing and were only dehydrated during freezing. All parts of the thawed cells were rehydrated. Ice crystal damage was mostly present in frozen mitochondria and axonemes and the acrosomes were strongly affected by thawing. The total amounts of Na, Cl, Ca, K, Mg, and Zn per cell were only elevated in frozen and thawed midpieces while the heads were permeable both to water and elements at that time.

Keywords: boar; spermatozoa; freezing; water; element concentrations; electron microscopy; image analysis; X-ray spectrophotometry

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B. P. Setchell, L. Plöen and E. M. Ritzen

The effect of two class III antiarrhythmic drugs (Almokalant, Astra-Hässle and Dofetilide, Pfizer) on fluid secretion by rat testes has been examined. Both drugs reduced fluid secretion, whether this was measured by the amount of rete testis fluid that could be collected 22 h after unilateral efferent duct ligation, or by the difference in mass between the ligated and unligated testes, or by the difference in amount of supernatant fluid after the parenchyma of the ligated and unligated testes had been dispersed and centrifuged. The secretion of potassium, calculated from the amount of potassium in the supernatant fluids from the ligated and unligated testes was also reduced by the drugs, whereas the secretion of androgen-binding protein and inositol was unaffected. The concentration of potassium in the secreted fluid, calculated from the amount and composition of the supernatant fluids, was not affected by treatment of the rats with Almokalant, but was increased in rats treated with Dofetilide and, in these, the concentration of sodium was reduced and that of magnesium and inositol was increased and the concentration of total protein was unaffected. The concentration of androgen-binding protein in secreted fluid was increased in rats treated with Almokalant, while the concentration of testosterone was unaffected. Histological examination of testes from treated rats revealed phagocytosis of stage 19 spermatids in tubules at stages VIII–IX after 2 days, at stages IX–XI after 4 days and at stages VIII–XIV after 7 days, apparently owing to an effect on spermiation. It appears that these drugs interfere with potassium-mediated fluid secretion by the testis, leading to the other changes seen.