The effect of age on sperm stock and egg laying in the parasitoid wasp, Dinarmus basalis.

Sperm quantity and quality during storage may be constraints acting on female fecundity and hence fitness. In Hymenoptera, the importance of sperm quality has rarely been considered, despite its central role in reproductive strategies and especially in sex ratio control. In these insects, fertilized eggs develop into females and unfertilized eggs into males. Experiments were conducted on the female wasp, Dinarmus basalis, in the laboratory with and without egg-laying resources (hosts). The first point was to test if sperm age influenced sperm storage by measuring sperm count and viability using a sperm viability test (SYBR14 : propidium iodide). The second point was the influence of prolonged storage in the female genital tract on the quantity, sex ratio and fitness of offspring produced. Results show that sperm viability in the spermatheca does not change significantly with maternal age, and that the sperm stock is not affected when females are deprived of hosts. Egg-laying is gradually restored after 21 days of host deprivation but remains at a low level after 115 days. The fitness of mated D. basalis females is therefore not constrained by sperm quantity or quality and seems to depend on host availability and female age.

Offspring and sex ratio are independent of sperm management in Eupelmus orientalis females.

Sperm stocks in both males and females of the parthenogenetic wasp Eupelmus orientalis were investigated at various points during reproduction and compared to the progeny of females in controlled conditions. One day-old virgin males had approximately 5500 sperm, and from a total of about 1697 sperm transferred per copulation, 21% are stored in the spermathecae by females 24 hours after mating. At the end of the egg-laying period (at least 42 days), 2/5 of the initial amount of sperm remained in this storage organ. This decrease (from approximately 350 to 150) occurred essentially during the first 21 days of egg-laying activity, indicating that the majority of sperm stored were used during this period. Between 21 days and the end of fertile life, the number of sperm remained constant. The mean offspring production throughout reproductive life after one mating was 153, with 56.5% of the daughters laid at the beginning of the laying activity. Sex ratio was entirely female biased during the first 15 days (mean=0.65), then it decreased and became nearly equal after 20 days. Present results propose that females maximize the production of daughters i.e. of inseminated eggs until the 20th day and after this time lay as many daughters and sons despite their still having stored sperm. Physiological constraints due to ageing are proposed to explain this phenomenon.

Axonemal tubulin polyglycylation probed with two monoclonal antibodies: widespread evolutionary distribution, appearance during spermatozoan maturation and possible function in motility.

Two monoclonal antibodies, AXO 49 and TAP 952, probed with carboxy-terminal peptides from Paramecium axonemal tubulin and with polyglycylated synthetic peptides, are found to recognize differently tubulin polyglycylation, the most recently identified posttranslational modification discovered in Paramecium axonemal tubulin. With these antibodies, we show that tubulin polyglycylation is widely distributed in organisms ranging from ciliated protozoa to mammals; it arose early in the course of evolution, but seems to be absent in primitive protozoa such as the Euglenozoa. Tubulin polyglycylation is the last posttranslational modification which takes place in the course of Drosophila spermatogenesis and its occurrence corresponds to the end of spermatozoan maturation. An involvement of polyglycylated tubulin in axoneme motility is suggested since AXO 49 and TAP 952 specifically inhibit the reactivated motility of sea urchin spermatozoa.

A massive new posttranslational modification occurs on axonemal tubulin at the final step of spermatogenesis in Drosophila.

Using two antibodies raised against Paramecium axonemal tubulin, a monoclonal antibody, AXO 49 (Callen et al., Biol. Cell 81, 95-119 (1994)), and a polyclonal antibody, PAT (Cohen et al., Biol. Cell 44, 35-44 (1982)), which have been shown elsewhere to detect a new posttranslational modification of tubulin presumably corresponding to polyglycylation, we have analyzed the occurrence of this modification during spermatogenesis in Drosophila. Results obtained by immunofluorescence on cysts isolated by laceration of testes showed that the antibodies reacted on axonemal microtubules of several species within the genus. Observation of different stages of differentiation of D. obscura sperm cells indicated, first, that the epitopes reactive with both antibodies appeared at late stages, and secondly, that they were detected simultaneously along all axonemes within a cyst. Immunofluorescence on semithin sections and electron microscopic immunocytochemistry on ultrathin sections confirmed that the appearance of the epitope recognized by the monoclonal antibody occurred at the time of the individualization process of spermatids in D. melanogaster. These results indicate that the posttranslational modification occurs as a very late event, after complete assembly of axonemal microtubules, and that the axonemal tubulin becomes modified when axonemal microtubules become coupled with the membrane, suggesting that the modification may in some way be induced by the microtubule-membrane interaction.

[Conservative treatment of sub-clinical noninfiltrating cancers of the breast (102 EOA in situ, TONO)]. / Le traitement conservateur des cancers du sein infra-cliniques canalaires non infiltrants (102 EOA in situ, TONO).

102 patients with sub-clinical intra ductal non invasive breast cancer (T0N0) treated by limited surgery and curative radiation therapy. Follow-up ranged from 2 to 10 years with a median follow-up of 59 months. The long term survival rate of this therapeutic approach, consisting of simple excision without any adjuvant treatment and radical mastectomy was close to 100%. The actuarial local recurrence rate was only 8.6% at 10 years (confidence interval: 4.6 to 12.6%), which strongly suggests that radiation therapy is active on multicentric foci. Salvage surgery could be performed in every case of local recurrence. The survival rate of in situ breast cancer (T0N0) treated by conservative radio-surgery is comparable to that of radical mastectomy, while cosmetic results and psychological impact appear to be better for the conservative technique (breast preservation rate = 91 to 96%).

Another way of being anisogamous in Drosophila subgenus species: giant sperm, one-to-one gamete ratio, and high zygote provisioning.

It is generally assumed that sexes in animals have arisen from a productivity versus provisioning conflict; males are those individuals producing gametes necessarily small, in excess, and individually bereft of all paternity assurance. A 1- to 2-cm sperm, 5-10 times as long as the male body, might therefore appear an evolutionary paradox. As a matter of fact, species of Drosophila of the Drosophila subgenus differ from those of other subgenera by producing exclusively sperm of that sort. We report counts of such giant costly sperm in Drosophila littoralis and Drosophila hydei females, indicating that they are offered in exceedingly small amounts, tending to a one-to-one gamete ratio after a single mating. As a result, most of them are successfully involved in a fertilization. Hence, the concept of "paternity assurance of individual sperm" arises. Evidence is further provided here that almost the entire sperm is incorporated into the egg during fertilization. Labeling with specific antibodies in fertilized eggs reveals intact axonemes up to late gastrulation. The question, then, is why selection has favored such an unusual strategy. Explanations related to some prefertilization functions are ruled out. It is therefore tentatively proposed that virtually every giant sperm constitutes a "direct paternal legacy to the embryo," which, in contrast to any male-derived nuptial gift, cannot be minimized by female remating. We suggest that dramatic shortage of giant sperm with a high prospect of fusion and increased zygote provisioning is merely another way of being anisogamous.

The reproductive incompatibility system in Drosophila simulans: DAPI-staining analysis of the Wolbachia symbionts in sperm cysts.

Any hitherto available technique used to detect the presence of Wolbachia, a rickettsia-like endosymbiont of Drosophila simulans, i.e., crossing with tester strains, electron microscope observations, DAPI-staining of embryo, or amplification of specific sequences by PCR are notably time-consuming techniques. Staining sperm cysts with the fluorochrome DAPI is here shown to be a fast and reliable technique to detect the symbiont and evaluate the level of infection of individual males. Wolbachia cells are abundant in sperm cysts and make up a mass which is eliminated in the waste bag during spermatogenesis. Such a mass was absent in all those strains known to be aposymbiotic. In infected strains, tetracycline treatment led to the disappearance of the symbionts within sperm cysts. In aging males, a decrease in the proportion of infected cysts were observed. There was however no apparent change in the number of symbionts in the cysts which remained infected. The reduction of reproductive incompatibility, which is observed in older males, may be explained by this process, i.e., a decrease in the frequency of infected cysts.

Rapid three-dimensional reconstruction at the light microscopic level and a technique for re-embedding the same semithin sections for electron microscopic examination.

Rapid three-dimensional reconstruction of serial sections at the light microscopic and ultrastructural levels was accomplished using a two-step technique. Fixed specimens were embedded in Epon and 1 microns sections were cut and placed on glass slides. One of every four sections was drawn onto transparency film for rapid three-dimensional reconstruction. The semi-thin sections were re-embedded in Epon and sectioned at 90 nm for examination in the electron microscopy.

Inoperable recurrence after breast-conserving surgical treatment and radiotherapy.

Factors associated with inoperable local recurrence were investigated by a clinical and pathologic review of 596 patients with Stages I and II carcinoma of the breast treated by breast-conserving operations and megavoltage radiotherapy. After a median follow-up period of 71 months, 13 of 70 local recurrences observed were anatomically unsuitable for salvage surgical treatment, affecting 2.2 per cent of patients initially treated. In most, inoperable recurrences clinically resembled primary inflammatory carcinoma of the breast. All inoperable failures occurred in patients with invasive ductal carcinoma and were associated with the presence of unfavorable prognostic features (positive nodes, histologic grade 3, negative estrogen receptor, vascular invasion and lymphocytic stromal reaction). Despite doxorubicin-based chemotherapy, prognosis after inoperable recurrence was quite poor, although survival time in excess of two years was seen in receptor positive, lower grade recurrent tumors in which treatment included hormone therapy.

[Reevaluation of indications for adjuvant hormone therapy in primary breast cancer with high metastatic risk]. / Réévaluation des indications d'une endocrino-hormonothérapie adjuvante dans les cancers primitifs du sein à haut risque métastatique.

This study concerns the correlation between ER and PR status, menopausal status and the effect of adjuvant hormonotherapy in high risk primary breast cancer patients. We have compared the results obtained in a randomized trial (Institut Paoli-Calmettes, Marseille) with those of a historic series (Centre René Huguenin, Saint-Cloud). The patients presented the same clinical and histological criteria and received identical therapeutic protocols (chemotherapy and/or hormonotherapy). Compared with patients receiving no adjuvant treatment, it appeared that: 1) in post-menopausal patients, a significant detrimental effect of tamoxifen was found in ER-negative patients, while in ER-positive patients hormonal treatment was well correlated with both the presence and level of steroid receptors; 2) in pre-menopausal patients, hormonal therapy (oophorectomy + tamoxifen) appeared to be mediated by a complex mechanism involving more than an ER-positive cell population. In the light of the published results, the present findings underline the importance of reevaluating the indications of hormonotherapy in terms of hormone receptors and menopausal status. They also indicate the importance of biological factors in the evaluation of response to therapy. They can identify, subsets of patients in whom a given therapeutic protocol is detrimental, even though it may be beneficial for the overall population. heterogeneity in response to therapy among patient subsets is one of the most important problems which confronts medical statisticians and clinical investigators.

Comparative kinetics of short and long sperm in sperm dimorphic Drosophila species.

All species of the Drosophila obscura group exhibit within-ejaculate sperm length dimorphism. The present work is a contribution to the understanding of sperm competition through a comparative study of sperm kinetic parameters in four of these species. Videomicrographic observations at 200 frames per second of sperm from males and females, out of the storage organ, prior or after storage were made. Drosophila sperm display both major and minor waves. The former is analysed by measuring coiling diameter (micron) and the latter by recording both beat frequency (s-1) and wave propagation velocity (micron.s-1). Results show that the 'behaviour' of short and long spermatozoa noticeably differ: short sperm kinetics remains unaltered after storage while both major and minor waves of long spermatozoa are markedly modified. Thus, evidence is provided here of a sort of "differential activation" which is assumed to result in different survival abilities of short and long sperm within the storage organ of females.

Can we predict the mating pattern of Drosophila females from the sperm length distribution in males?

In order to test the validity of the prediction of the mating pattern of females from the sperm length distribution in males, three species of Drosophila were analysed. Males in the three species are equally polygynous but females differ in the level of polyandry. A 'low recurrence polyandry' is observed in the sperm dimorphic species D. affinis while a 'high recurrence polyandry' is observed in the sperm monomorphic species D. latifasciaeformis and D. littoralis. These results are consistent with the hypothesis proposed previously that sperm dimorphism in males can only be maintained by a selective alternative in females (i.e. facultative female polygamy), whereas a stricter mating system (e.g. 'obligatory' polyandry) should only result in sperm monomorphism irrespective of the absolute value of sperm length.

Is breast conservation after local recurrence feasible?

The feasibility of conservative salvage surgery was addressed in a clinicopathologic study of the results of wide excision for 50 selected parenchymal intramammary recurrences after standard breast conserving treatment. After median follow-up of 51 months, 16 (32%) second local failures were observed (5-year local control 62%). Cox multivariate analysis of 18 parameters indicated that only disease-free interval and resection margins significantly influenced local control. 5-year local control was 92% for recurrences occurring after 5 years vs. 49% for shorter intervals, and 73% for negative vs. 36% for positive or indeterminate margins. Local control appeared independent of morphologic features, initial tumour stage, patient age, recurrent tumour size and location. Median survival after second local failure was 33 months; tertiary therapy obtained ultimate local-regional control in 8 of 16 cases. The authors conclude that wide excision is a particularly satisfactory alternative to salvage mastectomy for late recurrences. Negative margins are essential. Further study will be required to establish additional guidelines allowing improved patient selection.

Breast-conserving therapy for macroscopically multiple cancers.

Of 586 unilateral stage I-II breast cancers treated with conservative surgery and radiotherapy, 61 patients were found to have two or more macroscopic tumor nodules, diagnosed either clinically (n = 20), mammographically (n = 2), or on gross pathologic examination (n = 39). After a median follow-up of 71 months, 15 of 61 (25%) of the patients with multiple tumors developed recurrence in the treated breast, compared to 56 of 525 (11%) of patients with single cancers (p less than 0.005). Local failure occurred in 6 of 37 (16%) of bifocal tumors and in 9 of 24 (35%) of patients with 3 or more tumor foci. Recurrence was more frequent for multiplicity diagnosed clinically or mammographically (8 of 22 patients, 36%) than when it was apparent only to the pathologist (7 of 39 patients, 18%). Only 1 of 21 bifocal tumors diagnosed on gross examination recurred. Local failure occurred in only 1 of 22 cases with clearly negative resection margins; the remaining recurrences were associated with positive (n = 3) or indeterminate margins (n = 11). In contrast with recurrences of unifocal breast cancers, local failures in these patients tended to be located at a distance from the original foci, to be multifocal, or to be diffuse, including skin involvement. Only four recurrences presented as a single focus in the vicinity of the original primary tumors. This study indicates that macroscopically multiple breast cancers are at higher local failure risk, especially if multiplicity is clinically apparent, or if three or more gross nodules are seen on pathologic examination. Negative resection margins appear to be essential for satisfactory results.

Why are local recurrences after breast-conserving therapy more frequent in younger patients?

The influence of patient age on risk of recurrence in the breast was retrospectively studied in 496 stage I-II invasive ductal carcinomas treated by macroscopically complete primary tumor excision followed by radiotherapy. With a median follow-up of 71 months, local recurrence occurred in 13 of 62 (21%) patients younger than 40 years, compared with 48 of 434 (11%) older patients (P less than .025). Cox multivariate analysis of 18 parameters identified four that significantly determined risk: major lymphocytic stromal reaction (MCR), unsatisfactory resection margins, increasing histologic grade, and extensive intraductal cancer (DCIS) within the primary tumor. Compared with older patients, those younger than 40 years had tumors that more often exhibited MCR (36% v 20%, P less than .01), histologic grade 3 (42% v 28%, P less than .025), and very extensive DCIS (21% v 6%, P less than .001). The status of resection margins did not differ significantly between younger and older patients. Restriction of Cox analysis to patients younger than 40 indicated that risk was adequately described by MCR and percentage of DCIS, without consideration of grade or margins. For patients younger than 40, local failure occurred in four of five (80%) tumors with both MCR and more than 50% DCIS, in eight of 25 (32%) with either, and one of 32 (3.1%) with neither of these morphologic features. This study suggests that the higher local failure risk observed in patients younger than 40 years reflects the greater prevalence of certain morphologic characteristics in breast cancers in younger patients. Age itself does not appear to be an independent determinate of risk.

Risk factors for breast recurrence in premenopausal and postmenopausal patients with ductal cancers treated by conservation therapy.

Risk factors for local failure were evaluated for 496 clinical Stage I-II patients with infiltrating ductal carcinomas (median follow-up, 71 months) treated by conservative surgery and radiotherapy. Monofactorial analysis identified the following factors to be correlated with increased risk: moderate/marked mononuclear cell reaction (MCR), high histologic grade (G), extensive intraductal component (EIC), tumor necrosis, macroscopic multiplicity, estrogen receptor negativity, anatomic tumor size, age younger than 40 years, and vascular invasion. Only MCR, G, and EIC proved significant in Cox multivariate analysis. These risk factors were highly age dependent, with EIC markedly more prevalent in women younger than 50, MCR and G in women younger than 40. Separate Cox analysis for premenopausal patients showed that MCR/EIC determined risk independent of resection margins: tumors with MCR had a 28%, and with EIC a 22% probability of recurring locally by 5 years. Premenopausal patients with neither risk factor had a very low failure rate (2.6% at 5 years), regardless of age. For postmenopausal patients risk of breast recurrence was determined both by adequacy of resection margins and grade, with a high local failure rate for patients having G3 tumors with positive or indeterminate margins (31% at 5 years). The authors conclude that the microscopic examination is the only useful tool for assessing the risk of local failure, which is quite low for the majority of patients treated with breast conservation. High-risk patients can be recognized morphologically. The age dependence of morphologic risk factors appears to explain the high local failure rate seen in patients younger than 40.

The prognostic significance of late local recurrence after breast-conserving therapy.

Of 178 local recurrences occurring in 1593 patients with clinical Stages I-II breast cancer treated by conservative surgery and megavoltage radiotherapy, 71 were diagnosed after the 5th year. Compared with recurrences occurring prior to 60 months, late recurrences were less frequently inoperable (1/71, 1.4%, versus 18/107, 17%, p less than 0.001), were more often located at a distance from the initial primary tumor (23/71, 32%, versus 15/106, 14%, p less than 0.005), and had a more favorable prognosis (5-year survival 84% versus 61% for late and early operable recurrences, respectively, p = 0.05). Five-year metastasis-free survival after late failure depended mainly on the anatomic extent of the recurrence (87% for recurrences apparently confined to the breast versus 34% for relapses involving the axilla, p less than 0.002). Prognosis of late recurrence appeared to be unaffected both by location of the recurrence within the breast and by the type of salvage operation used (mastectomy versus wide excision). Local-regional control after salvage surgery was satisfactory (89% at 5 years). Whereas recurrence in the breast prior to 5 years profoundly affected survival after initial diagnosis, patients with late failure had identical 15-year survival as other 5-year survivors who never failed locally. Late recurrences were more frequent in patients younger than 40 at initial treatment, and in patients who had inadequate radiotherapy. We conclude that late local recurrences after breast conservation do not represent a serious management problem.

[Cathepsin D: an independent prognostic factor in cancer of the breast]. / La cathepsine-D: un facteur pronostique indépendant dans le cancer du sein.

The total amount of cathepsin-D (mature forms and pro-enzyme especially) in the cytosol of 88 breast cancers including 85 primary cancers and 11 axillary lymph node metastasis was measured by way of immuno-radiometric assay. Maximum follow-up is 58 months. Cathepsin-D was found to be independent of clinical and biological parameters including axillary lymph node involvement and oestradiol receptors (RE). Univariate analysis has pointed out a significant linkage between overall survival and cathepsin-D, using a cut-off level of 30 pmol/mg protein which is the most discriminating value. Cathepsin-D appeared to be particularly useful in lymph-node-positive and RE-negative patients but it was not significant in the node-negative population. Multivariate analysis of the overall survival (Cox model) revealed that it was strongly related to oestradiol receptor, lymph node involvement and cathepsin-D.