The whole meiotic process can occur in vitro in untransformed rat spermatogenic cells.

The aim of the present study was to assess whether the whole meiotic process of spermatogenic cells is able to take place in vitro. Fragments of seminiferous tubules from 20- to 22- or 28-day-old rats were seeded in medium containing 0.2% fetal calf serum in bicameral chambers and then cultured for 4 weeks in a chemically defined medium. The differentiation of meiotic germinal cells was followed by four criteria: (i) ultramicroscopic examination of the different types of germ cells present in the cell layer throughout the culture period; (ii) determination of the changes in DNA content per nucleus of the cell population seeded with time in culture; (iii) assessment of the ability of germinal cells to transcribe genes expressed after completion of meiosis; and (iv) monitoring the fate of BrdU-labeled leptotene spermatocytes. The ultrastructural study showed that the overall organization of the cells in the culture well recalls that of the seminiferous epithelium throughout the culture period. Moreover the identification of young round spermatids 21 days after seeding suggested that these spermatids had been formed very recently in culture. Determination of DNA content per nucleus showed that a 1C cell population could be observed after several days of cultures reaching 6 to 10% of total cells. An exponential-like increase in the amounts of the mRNAs encoding for TP1 or TP2 occurred from the time when 1C cells appeared in the culture until the end of the experiment. Finally, BrdU-labeled leptotene spermatocytes differentiated into pachytene spermatocytes and then into secondary spermatocytes, and BdrU-labeled round spermatids were observed from Day 21 of culture onward. Taken together these results indicate that the whole meiotic process from leptotene spermatocyte to round spermatid can indeed occur in vitro under the present culture conditions.

Meiotic differentiation of germinal cells in three-week cultures of whole cell population from rat seminiferous tubules.

The aim of the present study was to set up a culture system allowing most of the meiotic phase of rat spermatogenesis to occur in vitro. For that purpose, the differentiation of spermatogenic cells was monitored by three criteria: 1) examination of expression of genes specifically expressed at a high level in pachytene spermatocytes (the phosphoprotein p19 [p19] and the testis-specific histone TH2B) or in round spermatids (transition protein 1 [TP1] and transition protein 2 [TP2]) by reverse transcription-polymerase chain reaction (RT-PCR); 2) ploidy analysis; and 3) cytological and immunocytochemical study of the germ cells. In the first trial, we determined the changes in the ratios of p19:TP1 and TH2B:TP2 mRNA-related PCR products in the whole testis of rats between 18 and 60 days postpartum and related those results to the sequential appearance of the various types of spermatogenic cells during that period. In the second trial, our aim was to reproduce, in a culture system using seminiferous tubules from 23- to 25-day-old rats, the changes observed in vivo. The p19:TP1 and TH2B:TP2 ratios decreased dramatically in testicular extracts of rats between 32 and 40 days postpartum, i.e., at the time period during which round spermatids become more and more numerous in the testis. When seminiferous tubules were seeded in bicameral chambers, cell viability remained close to 70% of total cells throughout the 3-wk culture period. Both p19:TP1 and TH2B:TP2 ratios decreased during the first week of culture. This was attributable to a decrease in the levels of p19 and TH2B mRNAs and also to an enhancement in the relative amounts of TP1 and TP2. These changes were correlated with the appearance of a 1C cell population in the culture. Histological examination of the culture demonstrated that under the conditions of the present study, 5-bromo-2'-deoxyuridine-labeled pachytene spermatocytes of stages IV-VI were able to differentiate into secondary spermatocytes, then into round spermatids.

New staining methods for sperm evaluation estimated by microscopy and flow cytometry.

New staining methods and automated instruments are now available to evaluate the sperm cell in vitro. Individual compartments of the sperm cell, such as the nucleus and the plasma and acrosomal membranes, may be investigated, as well as the cell function as shown by mitochondria activity and capacitation. Various probes are used and they can be analyzed by direct light or fluorescent microscopy or by flow cytometry. The automated instruments allow objective and accurate analysis and quantification as well as the ability to evaluate large population of cells in a shorter time, thus providing accurate evaluation of sperm quality. However, before these test can be recommended for routine clinical and investigational use, in the stallion, they need to be confirmed on a larger number of stallions and their correlation with traditional semen parameters and with stallion fertility has to be demonstrated.

Use of free living stages to study the effects of thiabendazole, levamisole, pyrantel and ivermectin on the fine structure of Haemonchus contortus and Heligmosomoides polygyrus.

Ultrastructural changes induced in vitro by thiabendazole, levamisole, pyrantel and ivermectin in the free living larval stages of two trichostrongyles (Heligmosomoides polygyrus and Haemonchus contortus) were analysed. The observed damage for each anthelmintic is related to the known mode of action and compared to the damage commonly described in adults. The advantage of using larvae to study the effects of anthelmintics on the fine structure of nematodes rather than adults is described. Thiabendazole induced alteration of the cellular organization especially epithelial cells of the digestive tract. Changes in mitochondria were also seen. Levamisole caused contraction of muscle fibres whereas no specific lesions were observed with pyrantel. Ivermectin caused an hypertrophy of muscular groups. The interest of such a technique in research on the modes of action of anthelmintics is emphasized.

Characterisation of boar sperm dynein heavy chains by UV-vanadate dependent photocleavage.

Intact and Triton X-100 demembranated boar spermatozoa possess two main heavy chains with molecular masses (M(r)) of 430 and 460 kDa. These heavy chains were photo-cleaved within the axoneme under V1 conditions and produced two main fragments at 245 kDa and 185 kDa. Two minor fragments at 170 and 90 kDa were also obtained. In the presence of low Mg2+ (1 mM) a supplementary fragment of 200 kDa was also observed. The heavier chain was cleaved in the absence of ATP to give the 245 kDa fragment. The boar axonemal heavy chains cannot be directly extracted by high salt treatment of the demembranated sperm in presence of high level of protease inhibitors (HPI) but were extracted when the solution contained low protease inhibitor (LPI) concentrations. Electron microscopy showed that high salt treatment in presence of LPI extracted the outer arm mainly from the principal piece of the flagellum and less from the intermediate piece. Fractionation of the LPI high salt by chromatography or sucrose gradient allowed the obtention of a particle with ATPase activity, a size of 1.2 MDa and a sedimentation coefficient of about 20S. The particle was composed of two heavy chains of M(r) 320 and 340 kDa. These heavy chains can be photo-cleaved under V1 conditions and in absence of Mg2+. The sucrose gradient 20S fractions contained also two chains at 110 and 87 kDa which could be either intermediate chains or proteolytic fragments of the heavy chains. A chain at 63 kDa was also associated with the 20S fraction.(ABSTRACT TRUNCATED AT 250 WORDS)

O-chain expression in the rough Brucella melitensis strain B115: induction of O-polysaccharide-specific monoclonal antibodies and intracellular localization demonstrated by immunoelectron microscopy.

Spleen cells from mice infected with the rough Brucella melitensis strain B115 were fused with NSO myeloma cells. Hybridoma supernatants were screened in ELISA with cell walls (CW), sonicated cell extracts (CE) and rough lipopolysaccharide (R-LPS) of B. melitensis strain B115 and whole B. melitensis B115 cells. Surprisingly, 22 monoclonal antibodies (mAbs) reacting in ELISA with both CW and CE but not with R-LPS and bacterial cells were shown by immunoblot analysis and ELISA to react with smooth lipopolysaccharide (S-LPS). These mAbs also reacted in ELISA with O polysaccharides (OPS) from the smooth Brucella abortus strain 99 and the smooth B. melitensis strain 16M and thus recognize epitopes present on the O-chain. Proteinase K LPS preparations from B. melitensis B115 analysed by immunoblotting with one mAb (12G12) recognizing S-LPS of both A and M specificity displayed the typical S-LPS high-molecular-mass ladder pattern but no S-LPS was detected in the phenol/water/chloroform/light petroleum LPS preparation of the same strain. mAb 12G12, specific for S-LPS, and a mAb (A68/03F03/D05) specific for R-LPS were used to localize the O-chain and R-LPS expressed in B. melitensis strain B115 by immunoelectron microscopy. Immunogold labelling was observed at the surface of B. melitensis B115 cells with the anti-R-LPS mAb but not with the anti-S-LPS mAb. In ultrathin sections, immunogold labelling with the S-LPS specific mAb was observed in the cytoplasm and in the periphery of the cytoplasm, probably at the cytoplasmic membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromatin alterations induced by freeze-thawing influence the fertilizing ability of human sperm.

A previous cytochemical study revealed that chromatin alterations could be induced in frozen-thawed human sperm. In order to evaluate the biological consequences of such chromatin alterations, we evaluated the relationship between these chromatin alterations and the drastic decrease or loss of sperm fertilizing ability after freeze-thawing. Using acridine orange staining and Feulgen-DNA quantitative microspectrophotometry, the nuclear variables of sperm were compared using samples which had either recovered, or lost their fertilizing ability after freezing-thawing during artificial inseminations, despite good post-thaw motility. There was a clear decrease in Feulgen-DNA content and nuclear surface values in sperm which exhibited a loss of fertilizing ability. Thus freeze-thawing may alter the DNA/nuclear protein relationships and impair the fertilizing ability of human sperm.

Does in vitro capacitation alter chromatin stability of ejaculated human spermatozoa? Cytochemical studies.

In vitro capacitation of human spermatozoa is commonly evaluated by the progressive motility percent. However its effects on sperm chromatin have hardly been studied. Our aim was to determine the extent to which in vitro capacitation with two treatments (B2 or human follicular fluid) alters the chromatin of human spermatozoa, by using two analytical methods, acridine orange staining and Feulgen-DNA cytophotometric measures. Ejaculates were obtained from 23 men participating in our in vitro fertilization program, and several measurements were made on the same ejaculate for each subject. No alteration was observed for the percent of native DNA after capacitation in B2, but spermatozoa incubation during the same time in human follicular fluid was followed by a significant decrease of the percent of native DNA (P less than 0.01). Feulgen-DNA content significantly increased after capacitation in either B2 or follicular fluid (P less than 0.05, P less than 0.001 respectively), and so did sperm nuclear surface area (P less than 0.001). In this study we observed a negative correlation between Feulgen-DNA content and fertilization rate (P less than 0.02). Moreover, the greater effects on Feulgen-DNA content were observed in men with abnormal sperm, whose spontaneous percent of native DNA was lower (P less than 0.05) and Feulgen-DNA content higher (P less than 0.05) than in men with normal sperm. These results indicate that capacitation in B2 as well as in human follicular fluid may alter the chromatin stability of human spermatozoa. Such results suggest a partial decondensation state of human spermatozoa during in vitro capacitation. However, beyond some level of decondensation, the fertilizing ability could be altered.

Effects of freezing-thawing on the spermatozoon nucleus: a comparative chromatin cytophotometric study in the porcine and human species.

Freezing-thawing effects on the nuclei of porcine and human spermatozoa were studied by determining native DNA percentage from fluorescence after acridine orange (AO) staining and by analyzing chromatin structure by a quantitative microspectrophotometric study of Feulgen-DNA complexes before and after freezing. The study of boar spermatozoa revealed no alteration in native DNA percentage after freezing. However, native DNA percentage decreased significantly in human spermatozoa. Feulgen-DNA content and sperm nuclear surface area decreased in both species after freezing. These results prompted us to hypothesize an overcondensation of sperm chromatin after freezing-thawing. This overcondensation may be related to the lower conception rates obtained with human and porcine semen after cryostorage via defective decondensation.

Freezing and thawing alter chromatin stability of ejaculated human spermatozoa: fluorescence acridine orange staining and Feulgen-DNA cytophotometric studies.

Cryopreservation and freezing-thawing effects on the fertilizing ability of human spermatozoa commonly are evaluated by post-thaw motility. Various studies have depicted the ultrastructural changes caused by freezing-thawing, yet the chromatin alterations have been studied very limitedly. Our aim was to determine the extent to which freezing-thawing alters the chromatin of human spermatozoa, using two analytical methods: acridine orange staining and Feulgen-DNA cytophotometric studies. Both methods revealed a dramatic effect of freezing-thawing on sperm chromatin: the native DNA content decreased as did the Feulgen-DNA content, and sperm surface area was reduced. These results indicate an effect on DNA, diminished accessibility for Feulgen, and a decrease in nuclear surface area and prompt us to hypothesize a relationship between an "overcondensation" state for sperm chromatin after freezing-thawing and a lower conception rate for human semen after cryostorage.

Feulgen-DNA changes in the germ cells of the male vole (Microtus agrestis) during their development.

Consistent with changes in ploidy during germ cell maturation, the quantity of Feulgen DNA in round spermatids of voles is approximately a quarter of that in primary spermatocytes. The amount increases slightly in elongated spermatids, and then declines in spermatozoa from the testis, epididymis and vas deferens, below the level of round spermatids. Simultaneously nuclear area decreases so that in spermatozoa it is about one tenth that of primary spermatocytes. The rise in Feulgen DNA in elongated spermatids above the level found in round spermatids, and the decline in spermatozoa below this level, is probably caused by altered affinity of the desoxyribose for the leucofuchsin. This may be brought about by the complex physico-chemical changes occurring in chromatin and elsewhere in the nucleus and cell as they acquire their highly specialised final form.

DNA and protein changes in the spermatozoa of bulls treated orally with ethylene dibromide.

Oral treatment of bulls with ethylene dibromide caused a temporary reduction of the DNA and protein content and head area of epididymal and ejaculated spermatozoa.