RESUMEN

AIMS: The secretoglobin mammaglobin 1 (MGB1) is strongly expressed in breast tumours, and is therefore used to detect breast cancer metastases, although it has also been detected in other tissues. The aim of this study was to examine MGB1 expression and its hormonal regulation in human endometrium to further investigate the use of MGB1 as a marker molecule. METHODS AND RESULTS: Mammaglobin 1 expression was assessed immunohistochemically in endometrial samples from 60 normal fertile patients throughout the menstrual cycle, in 49 endometriotic tissue samples, in 15 endometrial adenocarcinomas, and in 36 breast carcinomas. In addition, 25 endometrial samples were analysed by western blot and quantitative real-time reverse transcription polymerase chain reaction. To prove hormonal regulation, primary endometrial epithelial cells were cultured with 17ß-oestradiol and promegestone. MGB1 was detected in human endometrial tissue, with peak expression during the luteal phase, in 31% of endometriotic samples, in 53% of endometrial adenocarcinomas, and in 64% of breast carcinomas. MGB1 mRNA expression was increased in vitro by hormonal treatment. CONCLUSIONS: Our data show that MGB1 expression is not restricted to normal and malignant breast tissue. Besides its documented occurrence in endometriotic and malignant endometrial tissues, MGB1 is also expressed in normal human endometrium, and such expression is controlled by steroid hormones.

Asunto(s)

Endometrio/metabolismo , Mamoglobina A/metabolismo , Biomarcadores/metabolismo , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Mama/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patología , Carcinoma Endometrioide/genética , Carcinoma Endometrioide/metabolismo , Carcinoma Endometrioide/patología , Endometriosis/genética , Endometriosis/metabolismo , Endometriosis/patología , Endometrio/anatomía & histología , Femenino , Humanos , Inmunohistoquímica , Mamoglobina A/genética , Ciclo Menstrual/genética , Ciclo Menstrual/metabolismo , Neoplasias Hormono-Dependientes/genética , Neoplasias Hormono-Dependientes/metabolismo , Neoplasias Hormono-Dependientes/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Distribución Tisular

RESUMEN

BACKGROUND: Class I histone deacetylases (HDACs) and acetylases (HATs) are members of transcriptional pre-initiation complexes assembled by steroid hormone receptors. Recently, HDAC inhibitors were shown to enhance differentiation of endometrial fibroblasts and endometrial adenocarcinomas. However, there is only rare information on HDAC and HAT expression in the human endometrium. METHODS: HDAC-1, -2, -3 and HAT (PCAF and GCN5) mRNA expression was studied in tissue from premenopausal women undergoing hysterectomy by real-time or semiquantitative RT-PCR. HDAC protein expression was assessed by Western Blot and immunohistochemistry. In endometrial adenocarcinomas (n = 17), HDAC-1 expression was studied by immunohistochemistry. RESULTS: In the human endometrium, HDAC-1, -2, -3 and PCAF mRNA are expressed without cyclical changes. Western blot analysis demonstrated that HDAC-2 protein expression was slightly, but significantly elevated in the secretory phase (P < 0.01 versus day 5-8), whereas HDAC-1 and -3 protein expression was constitutive throughout the menstrual cycle. By immunohistochemistry, nuclear expression of HDAC proteins was detected in all endometrial cell types. In the case of HDAC-3, immunostaining was significantly reduced in the endometrial surface epithelium on day 6-10 (P < 0.01 versus days 15-18 and 24-28). Compared to normal endometrium, a high proportion of endometrial adenocarcinomas showed impaired HDAC-1 protein expression in the epithelial and stromal compartment. CONCLUSIONS: Class I HDACs and HATs are expressed in the human endometrium throughout the menstrual cycle, suggesting the cyclic endometrium as a potential target for HDAC inhibitors. We hypothesis that alterations of HDAC and/or HAT expression are potentially involved in impaired endometrial differentiation.

Asunto(s)

Adenocarcinoma/enzimología , Neoplasias Endometriales/enzimología , Endometrio/enzimología , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Adulto , Femenino , Histona Desacetilasa 1 , Histona Desacetilasa 2 , Histona Desacetilasas/biosíntesis , Humanos , Inmunohistoquímica/métodos , Persona de Mediana Edad , Proteínas Represoras/biosíntesis , Útero/enzimología , Factores de Transcripción p300-CBP/biosíntesis

RESUMEN

OBJECTIVE: To study the mRNA expression of the two leucine-rich repeat-containing G-protein-coupled receptors LGR-4 and LGR-5 and the mRNA and protein expression of LGR-7, the relaxin receptor, in the human cyclic endometrium. DESIGN: Retrospective study. SETTING: Department of Anatomy and Reproductive Biology, Rheinisch-Westfälische Technische Hochschule Aachen, Aachen, Germany. METHOD(S): LGR-4, -5, and -7 mRNA expression was assessed by semiquantitative and real time reverse transcription-polymerase chain reaction in the endometrium of premenopausal women (n = 26) and cultured primary endometrial epithelial cells and fibroblasts (n = 3). Transcript size was determined by Northern blotting. LGR-7 protein expression was assessed by immunohistochemistry. RESULT(S): The mRNA of LGR-4, LGR-5, and LGR-7 was expressed constitutively throughout the menstrual cycle in the endometrium, and characterized by substantial differences in expression levels of individual women. LGR-7 immunostaining was detected in the epithelium of the functional layer throughout the cycle, with lowest staining in the midproliferative phase. Furthermore, individual stromal cells of the functional layer and the stroma of the basal layer showed LGR-7 immunostaining. CONCLUSION(S): Endometrial expression of the mRNA of orphan receptors LGR-4 and LGR-5 implies that the endometrium is potentially influenced by as yet unknown mediators, which are possibly involved in fertility control. Furthermore, we confirmed constitutive endometrial mRNA expression of LGR-7, the classical relaxin receptor, and demonstrated specific LGR-7 immunostaining of different endometrial cell types, which suggests a physiological role of relaxin in the human endometrium.

Asunto(s)

Endometrio/citología , Endometrio/fisiología , Proteínas de la Membrana/genética , Receptores Acoplados a Proteínas G/genética , Secuencia de Aminoácidos , Northern Blotting , Técnicas de Cultivo de Célula , Femenino , Humanos , Leucina , Ciclo Menstrual , Hibridación de Ácido Nucleico , Receptores de Péptidos , Valores de Referencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética , Útero/citología , Útero/fisiología

RESUMEN

OBJECTIVE: To examine immune cell phenotypes in viable tubal pregnancies (VTP) and in tubal abortions (TA). METHODS: Paraffin-embedded specimens of VTP (n=7) and ongoing TA (n=6) were double-stained for cytokeratin for trophoblast as well as for CD45, CD3, CD8, CD68 and CD20 for immune cell phenotypes. In all cases, the amniotic sac was detected by ultrasound. Histological examination showed no evidence of necrosis within the tissues included in this study. Quantification of the subpopulations was performed in each slide by two independent examiners in five areas (0.085 mm2 each) of the invasion zone as marked by cytokeratin-positive stromal extravillous trophoblast (EVT) cells. For statistical analysis, the non-parametric two-tailed t-test was used (p<0.05). RESULTS: The differences in the number of CD45(+), CD68(+) and CD20(+) cells was significant (p=0.0423, p=0.0469 and p=0.0494, respectively); however, the number of CD3(+), and among those the number of CD8(+) cells, was approximately eight-fold higher in TA than in VTP (p<0.0001 and p=0.0012, respectively). CONCLUSION: The unequal distribution of CD8(+) cells in VTP and TA suggests a significant role of this immune cell phenotype in the further outcome of a tubal pregnancy either to an abortive or a viable, potentially life-threatening, entity.

Asunto(s)

Aborto Espontáneo/inmunología , Amnios/inmunología , Linfocitos T CD8-positivos/inmunología , Trofoblastos/inmunología , Aborto Espontáneo/patología , Adulto , Amnios/ultraestructura , Antígenos CD/inmunología , Linfocitos T CD8-positivos/ultraestructura , Femenino , Humanos , Queratinas/inmunología , Embarazo , Trofoblastos/ultraestructura

RESUMEN

OBJECTIVE: To develop a method of freezing small amounts of spermatozoa in polymerized alginic acid drops, which can be liquified after thawing for recovery of the spermatozoa. DESIGN: Prospective clinical study. SETTING: Medical School, RWTH Aachen, Aachen Germany. PATIENT(S): None. INTERVENTION(S): Validation of the encapsulation method with bovine sperm; cryopreservation of human spermatozoa in alginic capsules. MAIN OUTCOME MEASURE(S): We optimized the cryopreservation method by testing different parameters influencing the freezing procedure, such as concentration of alginic acid, size of drops, time of polymerization, and culture media. RESULT(S): The final protocol was as follows: encapsulation by 7.3 mg/mL alginic acid forming 10-muL drops polymerized for 30 seconds and liquefied for 2.5 minutes in sodium citrate. Cryopreservation of human spermatozoa by this protocol resulted in a decreased motility of 18.3% compared with standard protocols but a 19.9% higher vitality of the immotile spermatozoa. CONCLUSION(S): No difference in viability of spermatozoa after both sperm-freezing procedures could be observed. Further investigation will be undertaken to reduce the amount of immotile but viable sperm after microencapsulation in alginic acid.

Asunto(s)

Alginatos , Materiales Biocompatibles , Criopreservación/métodos , Espermatozoides/citología , Animales , Cápsulas , Bovinos , Medios de Cultivo , Ácido Glucurónico , Ácidos Hexurónicos , Humanos , Masculino , Oligospermia/terapia , Estudios Prospectivos , Motilidad Espermática

RESUMEN

Impaired histone acetylation was recognized to be involved in carcinogenesis. Furthermore, histone deacetylase (HDAC) inhibitors induce differentiation of breast cancer cells and inhibit tumour growth. These results prompted us to study HDAC-1 and -3 expression in breast tumours to establish their potential therapeutic and prognostic significance. HDAC-1 und HDAC-3 protein expression was analyzed immunohistochemically on a tissue microarray (TMA) containing 600 core biopsies from 200 patients. HDAC-1 and -3 expression was correlated to steroid hormone receptor-, Her2/neu- and proliferation status of tumours as well as to overall and disease free survival. Moderate or strong nuclear immunoreactivity for HDAC-1 was observed in 39.8% and for HDAC-3 in 43.9% of breast carcinomas. HDAC-1 and -3 expression correlated significantly with oestrogen and progesterone receptor expression (both p< 0.001). HDAC-1 expression predicted significantly better disease free survival (DFS: p=0.044), in particular, in patients with small tumours of all differentiation types (DFS: p=0.016). Multivariate analysis demonstrated that HDAC-1 is an independent prognostic marker. Our data suggest that evaluation of HDAC-1 protein expression enables a more precise assessment of the prognosis of breast cancer patients. Thus, HDAC-1 expression analysis might be clinically useful to facilitate an individual, risk-directed, adjuvant systemic therapy in breast cancer patients.

Asunto(s)

Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/patología , Histona Desacetilasas/metabolismo , Análisis de Matrices Tisulares , Neoplasias de la Mama/mortalidad , Diferenciación Celular , Supervivencia sin Enfermedad , Femenino , Estudios de Seguimiento , Alemania/epidemiología , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Modelos de Riesgos Proporcionales , Tasa de Supervivencia

RESUMEN

Uteroglobin (UG) is a conserved protein which is induced by progesterone and secreted by the epithelia of various mammalian reproductive and respiratory organs. Recombinant bovine uteroglobin (recbUG), consisting of 80 amino acids with a C-terminal His6 tag, was overexpressed in Escherichia coli and purified. The protein was crystallized in two geometric forms, rhomboid and cuneate (wedge-shaped), by the hanging-drop vapour-diffusion method at 295 K. The rhomboid crystals diffracted to a maximum resolution of 1.6 A using synchrotron radiation. These crystals belong to space group P2(1)2(1)2, with unit-cell parameters a = 81.42, b = 82.82, c = 45.26 A, and contain four monomers per asymmetric unit. The cuneate crystals diffracted to 2.35 A resolution using a rotating-anode generator. These crystals belong to space group C222(1), with unit-cell parameters a = 43.39, b = 93.94, c = 77.30 A, and contain two molecules per asymmetric unit.

Asunto(s)

Uteroglobina/química , Animales , Bovinos , Cristalización , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Uteroglobina/genética , Uteroglobina/aislamiento & purificación , Uteroglobina/metabolismo

RESUMEN

OBJECTIVE: To compare cytokine expression profiles of decidua basalis (containing trophoblast cells) and decidua parietalis (without trophoblast cells) for determination of microenvironments in human first trimester decidua. DESIGN: Retrospective study. SETTING: School of Medicine, RWTH University of Aachen, Aachen Germany, and Bourgognekliniek Maastricht, Maastricht, The Netherlands. PATIENT(S): Forty-six women who had undergone elective first-trimester termination of viable pregnancy at 5 to 12 weeks. MAIN OUTCOME MEASURE(S): Quantitative cytokine protein analysis in decidual tissues by enzyme-linked immunosorbent assay, qualitative cytokine messenger (m)RNA analysis in isolated decidual cell samples, and comparative mRNA and protein analysis in tissues of decidua basalis compared with decidua parietalis. RESULT(S): Interleukin-2, interferon-gamma (Th-1), interleukin-4 (Th-2), and interleukin-1beta proteins are expressed in the human first-trimester decidua. Interleukin-2, interferon-gamma, and interleukin-4 mRNA mainly derive from the decidual tissue leukocytes. Interleukin-1beta mRNA is expressed by all decidual cell types. Interferon-gamma mRNA and protein is detected predominantly in the decidua basalis, which contains trophoblast cells. CONCLUSION(S): Microenvironments are established topographically by different expression of cytokines in decidua basalis and decidua parietalis. These locally specific patterns are indicative of fetomaternal cross-talk. Higher interferon-gamma concentrations in decidua basalis may influence leukocyte differentiation (e.g., macrophage activation) and trophoblast invasion (e.g., by induction of expression of major histocompatibility complex).

Asunto(s)

Citocinas/análisis , Decidua/inmunología , Trofoblastos/inmunología , Citocinas/genética , Femenino , Humanos , Inmunohistoquímica , Embarazo , Primer Trimestre del Embarazo , ARN Mensajero/análisis , Estudios Retrospectivos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

The process of implantation and trophoblast invasion is currently considered as the most limiting factor for the establishment of pregnancy. Molecular interactions at the embryo-maternal interface during the time of adhesion and subsequent invasion are crucial to the process of embryonic implantation. Both partners, the mother as well as the embryo, play equal roles in the embryo-maternal dialogue, the embryonic part being the main topic in this study. Investigations of the proteins in the extra-embryonic matrices (i.e. zona pellucida) indicate that the embryo participates intensively in this early embryo-maternal signalling. One unique feature during implantation process of primate embryos is the release of chorionic gonadotrophin, which seems to influence endometrial activity by two different mechanisms: (i) luteotrophic activity with increasing progesterone release and (ii) a direct action on the endometrium. Furthermore, embryonic interleukin-1beta may be involved in embryo-maternal signalling. Other significant signals in this interaction are most likely leukaemia inhibitory factor (LIF) and colony-stimulating factor (CSF), which stimulate matrix metalloproteinase (MMP)/insulin-like growth factor binding protein-1 (IGFBP-1) activity and the insulin-like growth factor (IGF) system, which is modulated by embryonic IGFBP-3. Similar significances are discussed for uteroglobin and haptoglobin. Finally, the phenomenon of maternal immunological tolerance, triggered by the presence of the early embryo, is fundamental to the understanding of implantation and trophoblast invasion. A tightly regulated balance between activated and inactivated T cells at the implantation site may control the beginning of adequate trophoblast invasion and also limit this invasion to a tolerable extent for the maternal system, consequently ensuring a biologically healthy haemo-chorial placenta.

Asunto(s)

Gonadotropina Coriónica/metabolismo , Implantación del Embrión , Interleucina-6 , Animales , Diferenciación Celular , Factores Estimulantes de Colonias/metabolismo , Citocinas/metabolismo , Embrión de Mamíferos/fisiología , Endometrio/patología , Femenino , Inhibidores de Crecimiento/metabolismo , Haptoglobinas/metabolismo , Humanos , Tolerancia Inmunológica , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Interleucina-1/metabolismo , Factor Inhibidor de Leucemia , Linfocinas/metabolismo , Modelos Biológicos , Modelos Moleculares , Placenta/fisiología , Embarazo , Unión Proteica , Estructura Terciaria de Proteína , Transducción de Señal , Somatomedinas/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Linfocitos T/fisiología , Factores de Tiempo , Uteroglobina/química

RESUMEN

Following attenuation of progesterone production corpora lutea are selectively cleared, a process associated with recruitment of macrophages. In the rabbit little is known about luteal immune cell phenotypes and expression of cytokines, which influence immune cells and resident luteal cells, during luteolysis. Consequently, we studied luteal immune cells by immunohistochemistry as well as luteal IL-10, TNFalpha, MCP-1, IFN-gamma, and IL-1beta mRNA expression by semiquantitative RT-PCR from day 8 to day 20 in pseudopregnant rabbits (d8-d20 p.hCG). Luteal function was assayed by serum progesterone levels. Functional luteolysis commenced by d14 p.hCG as indicated by attenuation of serum progesterone levels. X4(+) tissue macrophage levels increased transiently on d12 and d14 p.hCG, whereas CD5(+) T-cell levels transiently declined on these two days. CD68(+) macrophages increased progressively after d16 p.hCG. The luteal mRNA level of the anti-inflammatory cytokine IL-10 as well as the mRNA levels of the pro-inflammatory cytokines TNFalpha and MCP-1 increased after d16 p.hCG and remained elevated up to d20 p.hCG. IFN-gamma and IL-1beta mRNA expression did not vary systematically. In summary, luteolysis was associated with an initial transient increase of X4(+) macrophages and decrease of CD5(+) T-cells, and later recruitment of CD68(+) macrophages. During structural regression pro- and anti-inflammatory cytokines are upregulated possibly to control immune cell function.

Asunto(s)

Cuerpo Lúteo/metabolismo , Citocinas/genética , Luteólisis/metabolismo , Linfocitos/inmunología , Macrófagos/inmunología , Animales , Antígenos CD/análisis , Antígenos de Diferenciación Mielomonocítica/análisis , Antígenos CD4/análisis , Antígenos CD5/análisis , Antígenos CD8/análisis , Quimiocina CCL2/genética , Cuerpo Lúteo/inmunología , Factores de Crecimiento Endotelial/genética , Femenino , Expresión Génica , Granzimas , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intercelular/genética , Interferón gamma/genética , Interleucina-1/genética , Interleucina-10/genética , Linfocitos/citología , Linfocinas/genética , Macrófagos/citología , Progesterona/sangre , Seudoembarazo/inmunología , Seudoembarazo/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Conejos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Serina Endopeptidasas/genética , Factores de Tiempo , Factor de Necrosis Tumoral alfa/genética , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular

RESUMEN

Human development commences with a fertilisation cascade, initiated when the haploid chromosomes of the oocyte and the sperm unite, thereby forming a programme for the creation of a human being. During the first few days development proceeds to implantation and placentation in only 30% of cases. The frailty of this early development shows that a human blastocyst, should it manage to emerge from the zygote, will only succeed in turning into a human being if there is a maternal contribution to implantation and placenta formation. The obstacles to early development represent basic genetic and epigenetic phenomena within the cytoplasm and in the genome of the blastomeres and are the results of the operation of natural laws the exact causal concatenations of which remain yet to be determined in detail by science. Philosophers, ethicists and lawyers seem to have difficulties in accepting that more research in this field is required. Finally, a look at the legal protection of the human embryo in Germany reveals that the earliest stage of human development prior to implantation is legally protected in vitro, whereas there is no such legal protection in vivo, owing to Germany's abortion law (StGB (German Penal Code) section 218), before implantation. Aspects of the research on human embryos and embryonic stem cells are here discussed from this point of view.

Asunto(s)

Desarrollo Embrionario y Fetal/fisiología , Blastocisto/fisiología , Cromosomas Humanos , Implantación del Embrión/fisiología , Femenino , Humanos , Masculino , Placenta/fisiología , Embarazo , Interacciones Espermatozoide-Óvulo

RESUMEN

OBJECTIVE: To determine the localization and concentrations of estrogen receptor alpha and progesterone receptors A and B in the lower uterine segment during term parturition. METHODS: Biopsies were taken from 70 patients during nonelective cesarean delivery. The patients were at different stages of cervical dilatation (<2 cm, 2-3.9 cm, 4-6 cm, >6 cm) and different duration of labor (< or =6 hours, >6-12 hours, >12 hours). The receptor concentrations were determined with solid phase immunoassays, and their localization was investigated immunohistochemically. RESULTS: Estrogen receptor alpha concentration decreased significantly from 2.12 fmol/mg protein at less than 2 cm dilatation to 1.08 fmol/mg (4-6 cm) but tended to increase at greater than 6 cm. Progesterone receptor A and B concentration was 84.7 fmol/mg at less than 2 cm dilatation, decreased significantly to 36.6 fmol/mg (2-3.9 cm), and increased again with further dilation. Concentrations of both receptors did not depend on duration of labor. By immunohistochemistry only progesterone receptor A and B was found to be expressed by endothelial and smooth muscle cells of the vessels, stromal fibroblasts, smooth muscle cells in the myometrium, and glandular epithelial cells. Regardless of the extent of cervical dilatation, expression of progesterone receptors A and B was marked. CONCLUSION: A decrease in estrogen receptor alpha and progesterone receptor A and B concentration in the early phase of first stage labor may play a role in cervical dilation at term.

Asunto(s)

Trabajo de Parto , Receptores de Estrógenos/análisis , Receptores de Progesterona/análisis , Útero/química , Receptor alfa de Estrógeno , Femenino , Humanos , Inmunohistoquímica , Embarazo

RESUMEN

After its original description as a steroid-dependent protein in the rabbit uterus, uteroglobin became one of the best characterized proteins. However, detailed knowledge of its physiological role remains an enigma. In this study we investigate how its structure is phylogenetically conserved in the horse compared to other mammalian species. Northern blot analysis showed that in horses, the main expression of uteroglobin appears in lung, uterus, and prostate tissues. Western blot analysis demonstrated that the dimeric form of uteroglobin is found predominantly in biological compartments. Using a RACE-PCR technique, we cloned and sequenced the full-length cDNA (473 base pairs) that encodes equine uteroglobin. The nucleotide sequence was shown to characterize the primary structure of this protein. This enabled us to add equine uteroglobin to a comparative amino acid alignment of 8 other uteroglobin molecules, and finally, to unravel 14 evolutionary completely conserved amino acids. We summarize these results with a computer-based 3-D model of horse uteroglobin, and discuss new concepts on the physiological role of uteroglobin, in particular as a specific binding protein.

Asunto(s)

Secuencia de Aminoácidos , ADN Complementario/química , Caballos , Uteroglobina/química , Uteroglobina/genética , Animales , Secuencia de Bases , Northern Blotting , Western Blotting , Simulación por Computador , Secuencia Conservada , Evolución Molecular , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , Alineación de Secuencia , Análisis de Secuencia de ADN , Especificidad de la Especie