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Immunolocalization of bovine sperm protease BSp120 by light and electron microscopy during capacitation and the acrosome reaction: its role in in vitro fertilization.

Mammalian fertilization involves various steps in which the participation of specific enzymes has been demonstrated by numerous studies. Acrosin is one of the most widely acrosomal protease in mammalian spermatozoa studied, including bovine; however, other proteases have also been described. A new trypsin-like serine protease named bovine serine protease of 120 kDa (BSp120) and its pre-cursor BSp66 (66 kDa) were identified in bovine spermatozoa. Cytological and ultrastructural immunolocalization studies on BSp120 were performed in live and fixed cells. Immunoflorescence assays with specific polyclonal antibodies revealed localization of BSp120 on the sperm head, with a signal homogeneously distributed over the acrosome resembling a horseshoe. After the acrosome reaction, sperm showed a patchy pattern in the acrosomal cap. Immune electron microscopy analysis indicated that BSp120 is located over the head plasma membrane of capacitated spermatozoa and acrosome reacting spermatozoa. To assess BSp120 function in sperm-oocyte interaction, in vitro fertilization studies were conducted. Oocytes were incubated with spermatozoa pre-treated with anti-BSp120, anti-guinea pig acrosin, and anti-BSp120 plus anti-guinea pig acrosin. Pre-treatment of bovine spermatozoa with antibodies towards each protein did not significantly modify fertilization rates. However, when both anti-acrosin and anti-BSp120 antibodies were simultaneously added, there was a significant decrease in the fertilization rate, suggesting that both enzymes may be required for fertilization. Altogether, the results from the present study described the localization of BSp120 over the acrosome of bovine sperm, and suggest its involvement in fertilization.

Acrosome↗

Maternal chromatin remodeling during maturation and after fertilization in mouse oocytes.

Immunofluorescence staining with antibodies against acetylated histone H4 and 5-methylcytosine was carried out to investigate female chromatin remodeling throughout oocyte maturation and chromatin rearrangement involving both male and female genomes after fertilization. Oocyte cytoplasm remodels female chromatin in preparation of the fertilizing event and the subsequent chromatin rearrangement. Histone H4 are in fact progressively deacetylated whereas demethylating enzymes do not seem to be active over this period. The acetylase/deacetylase balance seems to be cell cycle dependent as female chromatin is deacetylated during maturation and reacetylated at telophase II stage both after fertilization and activation. On the contrary, DNA demethylation seems to be strictly selective. It is in fact confined to the remodeling of paternal genome after fertilization of mature oocytes as the ooplasm is not effective in demethylating either paternal chromatin in germinal vesicle breakdown (GVBD) fertilized oocytes or maternal genome of partenogenetically activated oocytes. Surprisingly, we induced maternal chromatin demethylation after fertilization by treating oocytes with a combination of a methyltransferase inhibitor, 5-azacytidine (5-AzaC), and a reversible and specific inhibitor of histone deacetylase, trichostatin A (TSA). This treatment likely induces a hyperacetylation of histones (thus favoring the access to demethylating enzymes by opening female chromatin structure) associated with a block of reparative methylation by inhibiting methytransferases. This manipulation of chromatin remodeling may have applications regarding the biological significance of aberrant DNA methylation.

Animals↗

Morphological abnormalities in the spermatozoa of fertile and infertile men.

The morphological analysis of the spermatozoa from fertile and infertile men was performed using light and electron microscopy to clarify the relationship between sperm morphology and fertility. Semen samples obtained from 22 partners of pregnant women were prepared according to the protocol standardized in an international collaborative study. Semen samples from 17 patients with asthenozoospermia or varicocele were collected in a hospital. Abnormalities in the spermatozoa were classified into three types for the tails, two for the midpieces, and six for the heads according to the criteria adapted from WHO guidelines (World Health Organization, 1999: WHO laboratory manual for the examination of human semen and semen-cervical mucus interaction (4th edition)). Approximately 14% of the spermatozoa from the fertile men had abnormal tails at the light microscopic level while approximately 44% had abnormal heads. Most types of abnormalities found in the spermatozoa from the asthenozoospermic and varicocele patients were encountered in those from the fertile men, although the semen from the fertile men contained a higher percentage of normal spermatozoa than that from the patients. These results were also confirmed at the ultrastructural level. Most abnormal cell types are encountered in semen from fertile men, although the incidence of abnormalities is low.

Case-Control Studies↗

The effect of cooling and hypertonic exposure on murine oocyte function, fertilization, and development.

Several individual but related steps are involved in the cryopreservation process, including the addition of cryoprotectants at various temperatures, cooling to subzero temperatures, and long-term storage. The process is completed by rewarming and removal of cryoprotectants prior to a return to physiological conditions. In this series of experiments we have attempted to distinguish the effects of some of these procedures. Control, untreated ovulated mouse oocytes showed 95% in vitro fertilization (190/200) and 92% subsequent development to hatching blastocyst (184/200). Exposure of oocytes to either isotonic or hypertonic media at 37 degrees C did not significantly change the rate of fertilization (90%, 108/120; and 89%, 154/174, respectively) or subsequent embryonic development (85%, 102/120; and 82%, 143/174, respectively). Slow cooling in isotonic medium (-3 degrees C/min) to 0 degree C had no effect on the rate of fertilization (83%, 103/124), but rapid cooling (> 1000 degrees C/min) to 0 degree C resulted in a significant reduction in fertilization rate to 75% (151/202). When oocytes suspended in a hypertonic solution were cooled using slow or rapid rates, there were marked decreases in fertilization to 26% (61/231) and 56% (156/278), respectively. Subsequent embryonic growth was reduced to 15% (34/231) after slow cooling and 26% (72/278) after rapid cooling. Exposure of oocytes to glycerol at 37 degrees C and dimethyl sulfoxide at 0 degree C reduced the fertilization rate to 57% (67/118) and 73% (103/145), respectively, with a corresponding reduction in embryonic growth to 52% (61/118) and 65% (94/145), but there were no additional effects of cooling or hypertonic exposure after addition of cryoprotectants.

Animals↗

Repetitive intracellular Ca2+ increases at fertilization and the role of Ca2+ in meiosis reinitiation from the first metaphase in oocytes of marine bivalves.

Spawned oocytes of marine bivalves Limaria hakodatensis, Mytilus edulis, Crassostrea gigas, and Hiatella flaccida are arrested at the first metaphase (metaphase-I) until fertilization. We have measured changes in intracellular Ca2+ ([Ca2+]i) at fertilization in the single oocytes of these bivalves using the fluorescent Ca2+ indicator fura-2. Shortly after insemination, these oocytes displayed a transient [Ca2+]i increase which was usually followed by a period during which [Ca2+]i was kept higher than the resting level (elevated [Ca2+]i period). During this period, [Ca2+]i showed oscillatory increases superimposed on an elevated [Ca2+]i level in Limaria, Crassostrea, and Hiatella, whereas a sustained elevation without pulses occurred in Mytilus. After [Ca2+]i returned to the resting level, repetitive transient [Ca2+]i increases appeared in Limaria, Mytilus, and Hiatella. The [Ca2+]i increases still occurred following external Ca2+ removal shortly after fertilization in all four bivalve species. In contrast, external Ca2+ removal immediately abolished a [Ca2+]i increase induced by excess-K+ seawater in Mytilus. Using another fluorescent Ca2+ indicator, calcium green, we found that during the first transient in Mytilus, [Ca2+]i increased uniformly over the whole oocyte. These results strongly suggest that the fashion of [Ca2+]i increases at fertilization in bivalve oocytes fertilized at metaphase-I differs not only from that in deuterostomes but also from that in protostomes oocytes of which are fertilized at the first prophase.

Animals↗

Competence to undergo normal, fertilization-induced cortical activation develops after metaphase I of meiosis in mouse oocytes.

The purpose of this study was to determine at what stage of meiotic maturation mouse oocytes develop the ability to undergo sperm-induced activation, assayed by cortical granule (CG) release. Germinal vesicle breakdown (GVBD), prometaphase I (proMI), metaphase I (MI), and metaphase II (MII, the normal stage at fertilization) stages were evaluated by quantitative image analysis. At 2 hr, fertilized MII eggs underwent a mean CG loss of 69%; loss was global--in the entire cortex occupied by CGs. In contrast, fertilized GVBD and proMI oocytes had no significant CG loss in the cortex after 2 hr. After 4 hr, 63, 67, and 88% of fertilized GVBD, proMI, and MI oocytes, respectively, had localized CG release only in the vicinity of the fertilizing sperm. At 4 hr, GVBD oocytes had small sperm-associated, CG-depleted domains, approximately 100 microns 2 (< 1% of the oocyte cortex), whereas those for proMI oocytes were approximately 2500 microns 2. The CG density was reduced by 90% in these domains, whereas the remaining cortex showed no evidence of CG loss. Both the extent and time course of CG loss were altered in oocytes fertilized before MII and were not significantly affected with 25 microM thimerosal, which sensitizes egg calcium stores. Unlike the ability to undergo CG release, the ability to initiate sperm chromatin decondensation was not stage specific and the extent of decondensation was cell cycle related. Competence to undergo CG loss develops in two phases: the ability to undergo localized CG release increases between the GV and MI stages, whereas the mechanism of propagating a normal wave of global CG loss from the site of sperm entry develops between MI and MII.

Animals↗

Effects of protein tyrosine kinase inhibitors on egg activation and fertilization-dependent protein tyrosine kinase activity.

Fertilization results in the activation of protein tyrosine kinases within minutes of sperm-egg binding, although the role of the kinase(s) involved is not clear. In the present study, we have treated sea urchin eggs with genistein, as well as other protein tyrosine kinase inhibitors, and have characterized the subsequent effect on fertilization and egg activation. Genistein treatment of sea urchin eggs inhibits the overall fertilization-dependent tyrosine kinase activity as well as the specific phosphorylation of a 350-kDa protein, but it did not inhibit cAMP-dependent kinase and had little effect on protein kinase C at concentrations less than 100 microM. Genistein, erbstatin, and tyrphostin B42 did not inhibit the early events of fertilization such as elevation of the fertilization envelope; however, later events such as pronuclear migration, DNA synthesis, and cell division were inhibited. These results suggest that protein tyrosine kinases activated following fertilization play a role in the later events of egg activation such as the initiation of pronuclear movement and entry into the S phase of the cell cycle.

Amino Acid Sequence↗

Protein synthesis increases after fertilization of sea urchin eggs in the absence of an increase in intracellular pH.

We have reevaluated the presumed requirement for an elevated intracellular pH (pHi) in the acceleration of protein synthesis which follows fertilization of eggs of the sea urchin Lytechinus pictus. Zygotes were transferred to sea water at a low pH (6.8) containing a permeant weak acid at times ranging from 5 min to as early as 30 sec postinsemination, to reverse or prevent the rise in pHi that normally ensues upon fertilization. Using the fluorescent pH probe 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF), we show that transfer of zygotes at 1 min or earlier after fertilization essentially blocks the rise in pHi. Under these conditions, both the incorporation of radiolabeled leucine into protein and the assembly of ribosomes into polysomes increase substantially (> 50% of control values). We also assessed leucine incorporation during incubation of eggs and zygotes in sodium-free sea water or sea water containing amiloride, two additional treatments that block the pHi rise. In the presence of amiloride, leucine incorporation increased upon fertilization, whereas little or no increase was observed in sodium-free sea water. We provide evidence that the low rates of leucine incorporation in sodium-free sea water result from the tendency for this experimental condition to lower the pHi to values significantly lower than the pHi in unfertilized eggs. These findings call into doubt the belief that the pHi rise at fertilization is a necessary prerequisite for the acceleration of bulk protein synthesis. These observations support the view that pHi is only one of several signals involved in the turn on of protein synthesis at the time of fertilization of sea urchin eggs.

Amiloride↗

Proteases stimulate fertilization-like responses in starfish eggs.

Application of proteases to eggs of the starfish, Asterina miniata, caused several responses like those seen at fertilization. Cortical granule exocytosis and fertilization envelope elevation occurred within about 1 min after exposure to trypsin, chymotrypsin, or pronase; protease inhibitors prevented these responses. Kallikrein caused cortical granule exocytosis and fertilization envelope elevation, but this response required more time (congruent to 30 min). Exocytosis was also seen in response to a recombinant trypsin, but not to a point-mutated trypsin without proteolytic activity. The extent of exocytosis was similar to that seen at fertilization, as measured by the fluorescent dye FM 1-43. In addition to causing exocytosis, application of trypsin, chymotrypsin, or pronase caused an increase in intracellular free calcium, detected by calcium green dextran, and stimulation of DNA synthesis, detected by incorporation of bromodeoxyuridine. Exocytosis also occurred when trypsin or chymotrypsin was applied in artificial sea water in which the free calcium was reduced to a low level (40-70 nM) such that Ca influx would be reduce by > 10,000-fold; this indicated that the proteases did not act by damaging the eggs and causing external calcium to leak into the cytoplasm. These findings show that there is an extracellularly exposed protein that when proteolyzed can induce fertilization-like responses; this protein may be a receptor that transduces a signal from the sperm to initiate egg activation at fertilization.

Animals↗

Repetitive calcium waves induced by fertilization in the nemertean worm Cerebratulus lacteus.

To analyze fertilization-induced calcium dynamics in a protostome worm, unfertilized oocytes of the nemertean Cerebratulus lacteus were co-injected with calcium green (CG) and rhodamine (Rh) dextrans for dual-channel confocal imaging of early development. Based on CG/Rh ratioed images collected every 800 msec, fertilization elicits a "cortical flash" of elevated free calcium that spreads rapidly around the oocyte without propagating as a point-source wave. A similar calcium transient occurs in unfertilized oocytes treated with KCl to depolarize the oolemma, and the fertilization-induced cortical flash is eliminated if cobalt is used to block calcium channels, collectively indicating that fertilization initially triggers an influx of calcium ions through voltage-gated calcium channels in the oolemma. However, within minutes after producing a cortical flash, C. lacteus oocytes begin to display a series of point-source, oscillating waves of elevated free calcium that are propagated at about 15 micron/sec. The first two calcium waves arise at the site of sperm fusion and typically fail to reach the antipode, but after sperm incorporation, the waves spread globally throughout the ooplasm and typically shift their origin to a pacemaker region in the vegetal cortex. About 10 oscillations with an average duration of 3.3 +/- 1.2 min are generated for approximately 60-100 min postfertilization as meiotic maturation is completed, and such waves continue to occur in cobalt-containing seawater or calcium-free seawater. Thus, wavelike calcium oscillations induced by fertilization are apparently dependent upon internal calcium stores, which in turn may contain IP3-insensitive and/or IP3-sensitive receptors based on experiments using ryanodine, caged IP3, and heparin. Unfertilized oocytes also display repetitive calcium waves following intracytoplasmic injections of whole sperm, and such oscillations are eliminated if the sperm suspensions are boiled prior to injection, suggesting the possible presence of a heat-labile sperm component that can elicit wavelike oscillations during fertilization.

Animals↗

Expression of inositol 1,4,5-trisphosphate receptors in mouse oocytes and early embryos: the type I isoform is upregulated in oocytes and downregulated after fertilization.

A fertilization-induced increase in intracellular Ca2+ is responsible for initiating all of the events of egg activation. In mammals, the Ca2+ increase takes the form of a series of Ca2+ oscillations showing complex temporal and spatial properties. To understand the nature of these changes, we have investigated the expression patterns of the three isoforms of the inositol trisphosphate receptor (InsP3R) during oocyte maturation and preimplantation development. We find that mouse oocytes express mRNAs for all three InsP3R subtypes. Semiquantitative ratio reverse-transcriptase polymerase chain reaction shows that the type II isoform is the predominant message in mature oocytes, representing 67% of the InsP3R mRNA. In contrast, protein analysis reveals that the type I isoform accounts for all of the detectable InsP3R protein, despite representing only 20% of the InsP3R mRNA. The levels of InsP3R protein were examined to determine whether they correlated with the Ca2+ signaling events surrounding the fertilization process. Type I InsP3R protein increased during oocyte maturation and, in addition, within 8 h of fertilization underwent a dramatic decrease. During development to the blastocyst the level of type I InsP3R protein did not return to prefertilization levels and types II and III remained below our detection limit. The decrease in InsP3R protein after fertilization was found to correlate with a decrease in the sensitivity of InsP3-induced Ca2+ release. These studies show that the expression of InsP3R mRNA is developmentally regulated, that Ca2+ signaling at fertilization is mediated exclusively through the type I InsP3R, and that the InsP3R is downregulated after fertilization.

Animals↗

Comparative biology of calcium signaling during fertilization and egg activation in animals.

During animal fertilizations, each oocyte or egg must produce a proper intracellular calcium signal for development to proceed normally. As a supplement to recent synopses of fertilization-induced calcium responses in mammals, this paper reviews the spatiotemporal properties of calcium signaling during fertilization and egg activation in marine invertebrates and compares these patterns with what has been reported for other animals. Based on the current database, fertilization causes most oocytes or eggs to generate multiple wavelike calcium oscillations that arise at least in part from the release of internal calcium stores sensitive to inositol 1,4,5-trisphosphate (IP3). Such calcium waves are modulated by upstream pathways involving oolemmal receptors and/or soluble sperm factors and in turn regulate calcium-sensitive targets required for subsequent development. Both "protostome" animals (e.g., mollusks, annelids, and arthropods) and "deuterostomes" (e.g., echinoderms and chordates) display fertilization-induced calcium waves, IP3-mediated calcium signaling, and the ability to use a combination of external calcium influx and internal calcium release. Such findings fail to support the dichotomy in calcium signaling modes that had previously been proposed for protostomes vs deuterostomes and instead suggest that various features of fertilization-induced calcium signals are widely shared throughout the animal kingdom.

Animals↗

Calcium release at fertilization of Xenopus eggs requires type I IP(3) receptors, but not SH2 domain-mediated activation of PLCgamma or G(q)-mediated activation of PLCbeta.

Elevation of intracellular Ca2+ at fertilization is essential for the initiation of development in the Xenopus egg, but the pathway between sperm-egg interaction and Ca2+ release from the egg's endoplasmic reticulum is not well understood. Here we show that injection of an inhibitory antibody against the type I IP(3) receptor reduces Ca2+ release at fertilization, indicating that the Ca2+ release requires IP(3). We then examine how IP(3) production is initiated. Xenopus eggs were injected with specific inhibitors of the activation of two phospholipase C isoforms, PLCgamma and PLCbeta. The Src-homology 2 (SH2) domains of PLCgamma were used to inhibit SH2-mediated activation of PLCgamma, and an antibody against G(q) family G-proteins was used to inhibit G(q)-mediated activation of PLCbeta. Though the PLCgamma SH2 domains inhibited platelet-derived growth factor (PDGF)-induced Ca2+ release in eggs with exogenously expressed PDGF receptors, they did not inhibit the Ca2+ rise at fertilization. Similarly, the G(q) family antibody blocked serotonin-induced Ca2+ release in eggs with exogenously expressed serotonin 2C receptors, but not the Ca2+ rise at fertilization. A mixture of PLCgamma SH2 domains and the G(q) antibody also did not inhibit the Ca2+ rise at fertilization. These results indicate that Ca2+ release at fertilization of Xenopus eggs requires type I IP(3)-gated Ca2+ channels, but not SH2 domain-mediated activation of PLCgamma or G(q)-mediated activation of PLCbeta.

Animals↗

Sperm-induced Ca(2+) oscillations in mouse oocytes and eggs can be mimicked by photolysis of caged inositol 1,4,5-trisphosphate: evidence to support a continuous low level production of inositol 1, 4,5-trisphosphate during mammalian fertilization.

During mouse fertilization the spermatozoon induces a series of low-frequency long-lasting Ca(2+) oscillations. It is generally accepted that these oscillations are due to Ca(2+) release through the inositol 1,4,5-trisphosphate (InsP(3)) receptor. However, InsP(3) microinjection does not mimic sperm-induced Ca(2+) oscillations, leading to the suggestion that the spermatozoon causes Ca(2+) release by sensitizing the InsP(3) receptor to basal levels of InsP(3). This contradicts recent evidence that the spermatozoon triggers Ca(2+) oscillations by introducing a phospholipase C or else an activator of phospholipase C. Here we show for the first time that sperm-induced Ca(2+) oscillations may be mimicked by the photolysis of caged InsP(3) in both mouse metaphase II eggs and germinal vesicle stage oocytes. Eggs, and also oocytes that had displayed spontaneous Ca(2+) oscillations, gave long-lasting Ca(2+) oscillations when fertilized or when caged InsP(3) was photolyzed. In contrast, oocytes that had shown no spontaneous Ca(2+) oscillations did not generate many oscillations when fertilized or following photolysis of caged InsP(3). Fertilization in eggs was most closely mimicked when InsP(3) was uncaged at relatively low amounts for extended periods. Here we observed an initial Ca(2+) transient with superimposed spikes, followed by a series of single transients with a low frequency; all characteristics of the Ca(2+) changes at fertilization. We therefore show that InsP(3) can mimic the distinctive pattern of Ca(2+) release in mammalian eggs at fertilization. It is proposed that a sperm Ca(2+)-releasing factor operates by generating a continuous small amount of InsP(3) over an extended period of time, consistent with the evidence for the involvement of a phospholipase C.

Animals↗

Egg activation at fertilization: where it all begins.

A centrally important factor in initiating egg activation at fertilization is a rise in free Ca(2+) in the egg cytosol. In echinoderm, ascidian, and vertebrate eggs, the Ca(2+) rise occurs as a result of inositol trisphosphate-mediated release of Ca(2+) from the endoplasmic reticulum. The release of Ca(2+) at fertilization in echinoderm and ascidian eggs requires SH2 domain-mediated activation of a Src family kinase (SFK) and phospholipase C (PLC)gamma. Though some evidence indicates that a SFK and PLC may also function at fertilization in vertebrate eggs, SH2 domain-mediated activation of PLC gamma appears not to be required. Much work has focused on identifying factors from sperm that initiate egg activation at fertilization, either as a result of sperm-egg contact or sperm-egg fusion. Current evidence from studies of ascidian and mammalian fertilization favors a fusion-mediated mechanism; this is supported by experiments indicating that injection of sperm extracts into eggs causes Ca(2+) release by the same pathway as fertilization.

Animals↗

Immunological identification of sperm antigens that participate in fertilization.

Using monoclonal antibodies (mAbs) as probes for specific components of the spermatozoon, we have initiated an investigation aimed at identifying those sperm components that participate in the events of gamete interaction. We intend to exploit these mAbs not only for identifying functional sperm components, but also for defining the constituents of discrete domains that comprise the structure of this highly differentiated cell. Among the large group of anti-sperm mAbs that we have generated, we have focused to date upon two categories. The first category consists of mAbs that localize to the acrosomal crescent, a restricted region of plasma membrane overlying the acrosome. Within this category, the mAbs share many similarities with regard to subclass, species and tissue cross-reactivity, and antigen solubility, in addition to cellular distribution. Nevertheless, despite these similarities, some mAbs in this category (e.g., M42) inhibit fertilization, whereas others (e.g., M41) are non-inhibitory. The block to fertilization observed in the presence of M42 is dependent upon the zona pellucida surrounding the egg. The specific event prevented by M42 appears to be the induction of the acrosome reaction at its physiological site, the surface of the zona pellucida. The sperm components recognized specifically by M42 are a cluster of high molecular weight moieties, ranging from approximately 220,000 to 240,000. The mAbs described in the second category display common localization at the equatorial segment of the sperm head. The pair of mAbs discussed from this category, M2 and M29, again bear considerable similarity to each other, yet differ significantly in their ability to inhibit fertilization. M2 does not inhibit, whereas M29 causes a marked inhibition of fertilization. With M29, however, the block to fertilization is independent of the zona pellucida. The M29 mAb interferes with sperm interaction with the egg plasma membrane subsequent to sperm attachment; since M29 does not prevent sperm binding to the egg plasma membrane, the specific event affacted, in all likelihood, is gamete membrane fusion. M29 recognizes a single sperm component, with subunit molecular weight of approx. 40,000. A variety of experiments are underway currently, both to characterize the antigens recognized by these mAbs further as well as to identify additional sperm components that participate in the fertilization process.

Acrosome↗

Selection of DNA sequences from interval 6 of the human Y chromosome with homology to a Y chromosomal fertility gene sequence of Drosophila hydei.

An experimental approach towards the molecular analysis of the male fertility function, located in interval 6 of the human Y chromosome, is presented. This approach is not based on the knowledge of any gene product but on the assumption that the functional DNA structure of male fertility genes, evolutionary conserved with their position on the Y chromosome, may contain an evolutionary conserved frame structure or at least conserved sequence elements. We tested this hypothesis by using dhMiF1, a fertility gene sequence of the Y chromosome of Drosophila hydei, as a screening probe on a pool of cloned human Y-DNA sequences. We were able to select 10 human Y-DNA sequences of which 7 could be mapped to Y interval 6 (the pY6H sequence family). Since the only fertility gene of the human Y chromosome is mapped to the same Y interval, our working hypothesis seems to be strongly supported. Most interesting in this respect is the isolation of the Y-specific repetitive pY6H65 sequence. The pY6H65 locus extends to a length of at least 300 kb in Y interval 6 and has a locus-specific repetitive sequence organization, reminiscent of the functional DNA structure of Y chromosomal fertility genes of Drosophila. We identified the simple sequence family (CA)n as one sequence element conserved between the Drosophila dhMiFi fertility gene sequence and the homologous human Y-DNA sequences.

Animals↗

Yield and content in nitrates, minerals and ascorbic acid of leeks and turnips grown under mineral or organic nitrogen fertilizations.

The influence of mineral NPK fertilizers and organic fertilizers such as manure compost, woodchip compost and blood meal on the yields and the nutritive value of leeks and turnips were compared. Fertilizers were applied on the two crops grown successively in 200-1 containers. Mineral fertilizers, manure compost and blood meal provided equivalent vegetable yields. Contents in dry matter, ascorbic acid and minerals of leeks and turnips were not strongly affected by the different fertilization regimes. The nitrate content of both crops was significantly lower under the application of manure compost and woodchip compost, while mineral fertilizers and blood meal induced similar, higher nitrate levels. The present work thus provides new data showing the usefulness of composts in growing vegetables with low nitrate contents.

Agriculture↗