Human fertilization: epididymal hCRISP1 mediates sperm-zona pellucida binding through its interaction with ZP3.

Human epididymal CRISP1 (hCRISP1) associates with sperm during maturation and participates in gamete fusion through egg complementary sites. Its homology with both rodent epididymal CRISP1 and CRISP4 reported to participate in the previous stage of sperm binding to the zona pellucida (ZP), led us to further investigate the functional role of hCRISP1 by studying its involvement in human sperm-ZP interaction. Human hemizona (HZ) were inseminated with human capacitated sperm in the presence of either anti-hCRISP1 polyclonal antibody to inhibit sperm hCRISP1, or bacterially-expressed hCRISP1 (rec-hCRISP1) to block putative hCRISP1 binding sites in the ZP. Results revealed that both anti-hCRISP1 and rec-hCRISP1 produced a significant inhibition in the number of sperm bound per HZ compared with the corresponding controls. The finding that neither anti-hCRISP1 nor rec-hCRISP1 affected capacitation-associated events (i.e. sperm motility, protein tyrosine phosphorylation or acrosome reaction) supports a specific inhibition at the sperm-egg interaction level. Moreover, immunofluorescence experiments using human ZP-intact eggs revealed the presence of complementary sites for hCRISP1 in the ZP. To identify the ligand of hCRISP1 in the ZP, human recombinant proteins ZP2, ZP3 and ZP4 expressed in insect cells were co-incubated with hCRISP1 and protein-protein interaction was analyzed by ELISA. Results revealed that rec-hCRISP1 mainly interacted with ZP3 in a dose-dependent and saturable manner, supporting the specificity of this interaction. Altogether, these results indicate that hCRISP1 is a multifunctional protein involved not only in sperm-egg fusion but also in the previous stage of sperm-ZP binding through its specific interaction with human ZP3.

Frecuencia de criptorquidia y factores asociados en recién nacidos

Objetivos: Determinar la frecuencia de las anormalidades del descenso testicular y los factores asociados en recién nacidos (RN). Métodos: Se realizó un estudio clínico observacional tipo caso control. Durante el período noviembre 2007- Agosto 2008, se examinaron en el Instituto Autónomo Hospital Universitario de los Andes (IAHULA) todos los RN con criptorquidia, los cuales se compararon con 105 RN sin criptorquidia (grupo control). Datos complementarios fueron obtenidos mediante una encuesta a los padres. Resultados: De un total de 2084 RN, 35 presentaron criptorquidia, lo cual corresponde a una frecuencia del 1,7%. La criptorquidia ocurrió en 10,8% de los RN pre-término y solamente en 0,8 % de los RN de término. La prematuridad y el bajo peso al nacer se asociaron con mayor frecuencia con la criptorquidia (p<0,05). Otros factores como la talla, el índice Apgar y la longitud del pene de los RN, también estuvieron asociados con la presencia de criptorquidia. El riesgo relativo indirecto (Odds ratio) de presentar criptorquidia es 5,27 veces mayor en un RN pre-término comparado con un RN de término. Los RN con malformaciones congénitas tienen un riesgo 7,03 veces mayor de tener criptorquidia que un RN sin malformaciones congénitas. Conclusiones: Se confirma que en nuestros niños la criptorquidia es más frecuente en RN pre-término y que la frecuencia de criptorquidia en RN es del 1,7%. El bajo peso al nacer, la prematuridad, el pene de pequeñas dimensiones junto con la presencia de anomalías congénitas asociadas, son factores que se relacionan con el mal descenso testicular.

Objectives: To establish the frequency of abnormalities of testicular descent and associated factors in newborn (NB) boys. Methods: An observational clinical study, case control type, was performed. All criptorchydic NB were evaluated at the Hospital Universitario de Los Andes, from November 2007 to August 2008. They were compared with 105 NB without cryptorchidism. A survey to obtain data from the patients, and their parents was conducted. Results: Criptorchidism was present in thirty five out of 2084 male newborns, representing 1.7% of the sample. In preterm NB, the frecuency of criptory was 10.8%, compared to 0.8% frequency observed in at term male newborns. Prematurity and low birth weight were associated with increased frequency of chryptorchidism (p<0,05). Newborns height, Apgar index, and the lenght of their penis, were also associated with increased presence of chryptorchidism. The calculated odds ratio risk for criptorchidism is 5.27 times higher in pre-term NB compared to term boys at birth. The risk for chriptorchidism in a newborns with morphological developmental abnormalities, is 7.03 times higher than in a NB without such abnormalities. Conclusions: We have verified that criptorchidism in our children is more frequent in pre-term newborns, and that 1.7% is the frequency of criptorchidism in this sample. Low birth weight, prematurity, and the small penis dimensions, together with associated morphological developmental abnormalities, are risk factors related to abnormal testicular descent.

Transcription of DNA templates associated with histone (H3 x H4)(2) tetramers.

To investigate the in vitro transcription by bacteriophage T7 RNA polymerase of oligonucleosomes lacking histone H2A x H2B dimers, templates were assembled from histone (H3 x H4)(2) tetramers with and without the complementary amount of H2A x H2B dimers and two different DNA species: pGEMEX-1, devoid of nucleosome positioning sequences, and T7-207-18, which contains downstream from the promoter 18 tandem repeats of a 207-bp positioning sequence. Assembly with core histone octamers affects pGEMEX-1 transcription mainly at the initiation level, while T7-207-18 is almost exclusively inhibited at the level of elongation. With both DNA templates and under different salt conditions, RNA synthesis is much more efficient on oligonucleosomes containing only (H3 x H4)(2) tetramers than on those with whole histone octamers. Under conditions promoting a low transcription rate, it is unambiguously shown with pGEMEX-1 that the block to initiation due to the presence of core histone octamers is substantially removed when (H3 x H4)(2) is substituted for the whole octamer. With T7-207-18, under assay conditions allowing transcription of the whole coding region of the naked DNA, analysis of the transcription products indicates that RNA elongation on the template containing (H3 x H4)(2) tetramers takes place as easily as on free DNA, in contrast with the significant inhibition observed in the presence of whole histone octamers.

Repressive effect on oligonucleosome transcription of the core histone tail domains.

Histone-DNA templates for bacteriophage T7 RNA polymerase were assembled from histone octamers and three different DNA species, two circular (pGEMEX-1 and pT207-18) and one linear (T7-207-18). pGEMEX is devoid of nucleosome positioning sequences, while in pT207-18 and T7-207-18 the region downstream of the promoter contains 18 tandem repeats of a 207 bp positioning sequence derived from the 5S RNA gene of the sea urchin Lytechinus variegatus. Elimination of the histone tails in the assembled oligonucleosomes by trypsin digestion is accompanied, in all three DNA species, by substantial increases in transcription efficiency, assayed at different KCl and MgCl2 concentrations, after allowing for the aggregation observed under certain conditions. In the absence of KCl and at low MgCl2 concentration, the presence of 2 mM spermidine causes substantial aggregation of the intact oligonucleosomes but has a much smaller effect on those trypsin digested. The untreated histone-DNA templates, assembled on pGEMEX-1 and T7-207-18, give transcription products significantly shorter than those obtained with the corresponding free DNA. With oligonucleosome templates lacking histone tails, the transcripts have an average length intermediate between those corresponding to free DNA and intact histone-DNA, which indicates a partial elimination of the elongation restrictions imposed by intact histone octamers. The absence of histone terminal domains facilitates both transcriptional initiation and elongation. Apparently, the interaction of the histone tails with DNA at the nucleosomal level is responsible, at least in part, for their repressive effect on transcription.

Presence of an unusually high concentration of an ubiquitinated histone-like protein in Trypanosoma cruzi.

The conjugation of ubiquitin to histones H2A and H2B has been established in higher eukaryotes and has been related to changes in chromatin organization. In Trypanosoma cruzi, no condensation of chromatin occurs during mitosis. In order to determine the presence of histone ubiquitination in T. cruzi epimastigotes, histones were extracted from chromatin and analyzed by three electrophoretic systems: acid-urea, triton-acid-urea and sodium-dodecyl-sulphate polyacrylamide gel. The immunochemical detection of ubiquitin-histone conjugates by Western blotting showed a strong reaction with a slow migrating band of M(r) 19 kDa. The high percentage of ubiquitin-histone conjugates present in T. cruzi chromatin may be related to the inability of this parasite to condense chromatin into a 30 nm fiber.