Gene transfer to the mammalian reproductive tract.

This review summarizes the results of research on gene transfer to the mammalian genital tract. Gene transfer experiments have been developed during the last 2 decades and have been applied using in vitro, ex vivo and in vivo procedures. (i) In vitro methods have been applied to the uterine epithelial cells with the principal purpose of analysing some pathological change occurring in the uterus. In the male tract, epididymal cell lines have been used to evaluate the expression of particular genes and the function of specific proteins. (ii) Ex vivo methods have been applied to both the uterus and the vas deferens in humans, and good transgene expression has been recorded. (iii) In vivo gene transfer in the female tract has been employed in the uterus and oviduct using gene injections or electroporation methods. The glandular epithelium of both organs can be transfected efficiently, and transfection efficiency depends on the hormonal stage of the animal. The best expression occurred during pseudopregnancy and meta-estrus periods, when high progesterone and low estradiol concentrations occur. In the male tract, in vivo methods have been applied to mouse vas deferens and epididymis. In both organs, patches of epithelial regions appeared to express the transgenes. Furthermore, the secretions of both organs were also modified using gene constructions that led to the expression of some secretory proteins. In summary, gene modifications in the epithelium of the mammalian reproductive tract have been successful employing different technologies. Further improvements in transfection efficiency would help provide new insights into the physiology of these reproductive organs. Furthermore, the use of these methods could also be used to modify the fertility of mammals.

In vitro transfection of the human vas deferens using DNA-liposome and DNA-neutral lipid complexes.

OBJECTIVE: To transfect the human vas deferens in vitro. DESIGN: Prospective study, description of a procedure. SETTING: Research center and university hospital. PATIENT(S): Seven fertile men undergoing vasectomies or vasovasostomies. INTERVENTION(S): Human vas deferens pieces were transfected in vitro using the p-GeneGrip gene construction, which codifies for the green fluorescent protein (GFP). Lipofectamine or GenePorter were employed as gene vectors. MAIN OUTCOME MEASURE(S): After vas deferens epithelium transfection, we described the vas deferens foreign gene expression. Maximum transfection occurred in 14.7% of the vas deferens epithelial cells. After using GenePorter, we observed green fluorescent protein gene expression in 40% of samples, which occupied 9.86% of the epithelial area. After Lipofectamine treatment, transgene expression occurred in 33% of the samples and occupied 9.05% of the epithelial area. CONCLUSION(S): The human vas deferens epithelium has the potential to be modified by gene transfection.

Cohesin is needed for bipolar mitosis in human cells.

Multi-polar mitosis is strongly linked with aggressive cancers and it is a histological diagnostic of tumor-grade. However, factors that cause chromosomes to segregate to more than two spindle poles are not well understood. Here we show that cohesins Rad21, Smc1 and Smc3 are required for bipolar mitosis in human cells. After Rad21 depletion, chromosomes align at the metaphase plate and bipolar spindles assemble in most cases, but in anaphase the separated chromatids segregate to multiple poles. Time-lapse microscopy revealed that the spindle poles often become split in Rad21-depleted metaphase cells. Interestingly, exogenous expression of non-cleavable Rad21 results in multi-polar anaphase. Since cohesins are present at the spindle poles in mitosis, these data are consistent with a non-chromosomal function of cohesin.

Rad21 is required for centrosome integrity in human cells independently of its role in chromosome cohesion.

Classically, chromosomal functions in DNA repair and sister chromatid association have been assigned to the cohesin proteins. More recent studies have provided evidence that cohesins also localize to the centrosomes, which organize the bipolar spindle during mitosis. Depletion of cohesin proteins is associated with multi-polar mitosis in which spindle pole integrity is compromised. However, the spindle pole defects after cohesin depletion could be an indirect consequence of a chromosomal cohesion defect which might impact centrosome integrity via alterations to the spindle microtubule network. Here we show that the cohesin Rad21 is required for centrosome integrity independently of its role as a chromosomal cohesin. Thus, Rad21 may promote accurate chromosome transmission not only by virtue of its function as a chromosomal cohesin, but also because it is required for centrosome function.

Photoperiod-induced apoptosis in the male genital tract epithelia of the golden hamster.

The aim of this study was to identify some details of the changes induced by a short-day light regime (8:16 light:dark) on the male genital tract and accessory sex glands of the golden hamster Mesocricetus auratus. We principally examined the presence of apoptotic cells in the epithelium from different regions of the epididymis, seminal vesicles, prostate and coagulating gland. We detected an increase in the percentage of apoptotic cells in situ using the TUNEL technique in animals that were maintained for 6, 8 or 12 weeks in a short photoperiod. That those cells were indeed undergoing apoptosis was confirmed by the immunodetection of the active fragment of caspase-3. The apoptotic indices in the different tissues analysed were low, but were maintained for weeks, suggesting cell loss at a steady rate. We tried to correlate these changes with the testosterone levels in serum as well as with the oxidative stress in the tissue. On the other hand, the increase in size and number of lipofuscin granules indicated the possibility that a parallel increase in oxidative stress occurred in the tissues. The normalization in the number of apoptotic cells and lipofuscin granules in animals treated with testosterone suggests that both phenomena might be related to changes in the hormone levels.

Transfection of spermatozoa in bivalve molluscs using naked DNA.

Spermatozoa from the bivalve molluscs Mytilus galloprovincialis, Mytilus chilensis and Chamelea gallina were transfected in vitro using the p-GeneGrip construct, which encodes green fluorescent protein. The efficiency of transfection after brief incubation was assessed by fluorescence and confocal laser microscopy, and was about 58.5-70.01% in the species used. The foreign gene was principally located in the sperm nuclei, as demonstrated by laser confocal serial sections. In some spermatozoa, mitochondria, which are grouped in the base of the nucleus, also appeared to be transfected. Polymerase chain reaction and Southern blot analyses suggested that the foreign DNA had been integrated into the nuclear genome in Mytilus galloprovincialis spermatozoa. This simple method for spermatozoon transfection in molluscs of commercial interest could have biotechnological applications.

Ageing-induced changes in the cortical granules of mouse eggs.

The cortical cytoplasm and cortical granules (CGs) of mouse oocytes were analysed by electron microscopy. Oocytes were collected soon and 20h after ovulation from adult young females (3-4 months old). In addition, gametes collected soon after ovulation from 12- to 14-month-old females were used. Ultrastructural analyses were undertaken using the conventional procedures and the alcoholic PTA method. PTA selectively stains the CGs indicating the presence of lysine-rich proteins in these granules. Oocytes from young females showed CGs as dense granules 300-500 nm in diameter linearly arranged under the oolemma. In oocytes recovered 20h after ovulation 24.31% of CGs appeared vacuolated and 38.40% internalized in the cytoplasm. In gametes collected from old females several changes were observed in the cortical cytoplasm: (a) CGs appeared concentrated in some areas while others regions were devoid of granules; (b) groups of CGs appeared internalized in the egg cytoplasm; (c) the CG contents had swollen and changed, showing dense and clear areas; (d) numerous dense structures and vesicles (lysosome-like vesicles) were present; (e) cytoplasmic fragmentation was frequently seen. Fragments contained CGs, dense structures and vacuoles. These changes are closely related to the low fertilization rates shown by these oocytes when they were used for in vitro fertilization procedures.

Abdominal temperature induces region-specific p53-independent apoptosis in the cauda epididymidis of the mouse.

It is widely accepted that temperature regulates gene expression and function in the epididymis. However, the significance of reduced temperature of the scrotum in cell survival had not often been examined. Our hypothesis was that the experimental increase of the temperature could induce apoptosis. Using a surgical method that consists of surgically reflecting the cauda epididymidis in the abdomen, we have been able to show that this is the case. Apoptosis was examined by histologic procedures and by visualization of DNA fragmentation in agarose gels. We determined that the apoptosis is region-specific and affects only the principal cells of the proximal region of the cauda. It starts 12 h after surgery and ends by the third day. The apoptotic cells are eliminated by extrusion into the lumen and phagocytosis by adjacent cells. The complete molecular mechanism of apoptosis in this case remains unknown, but we have used the techniques of immunocytochemistry, Western blot, and reverse transcription-polymerase chain reaction to determine the role of some molecules. We have seen no significant role of androgens, the tumor suppressor p53, nor two heat shock proteins, hsp-25 and hsp-70. Nevertheless, we have detected a strong induction of bax and bcl-2 gene products. While the former should be responsible for the apoptosis observed, the latter would promote the survival of most of the cells of the cauda epididymis.

Generation of transgenic mice by transfection of pronuclear embryos using lipid-DNA complexes.

We had previously developed a methodology to introduce foreign DNA into mouse eggs and embryos using cationic lipids as vectors. In this report we use this technique to produce transgenic animals. Mouse embryos at the pronuclear stage were transfected using a mixture of a plasmid DNA, encoding for a nuclear form of beta-galactosidase, and a commercial lipid transfection reagent. Embryos were washed and incubated overnight. Those that cleaved and develop to the 2-cell stage of normal appearance were transferred to the Fallopian tubes of pseudopregnant foster mothers. We analysed a total of 158 offspring and found two, a female and a male, to be transgenic (1.27% of the total). Integration of the foreign DNA in the female was showed by Southern blot. Both animals expressed the lacz in several organs, but none of them either displayed expression in the germ cells or transmitted the transgene to their offspring. Taken together our results show that lipid transfection can generate transgenic mice, but the efficiency needs to be improved for this method to be widely applied.

In vivo transfection of the mouse vas deferens.

To explore the possibility that specific characteristics of the epithelium of the male tract can be modified, transfections of the mouse vas deferens have been performed using in vivo injections of cationic DNA/liposome complexes. Gene transfer was done employing the reporter genes pEGFP-C1 encoding Green Fluorescent Protein (GFP) and pCMV-nls-beta encoding the nuclear beta-Galactosidase (beta-Gal). Foreign gene expression reached a maximum of 6.8% in the epithelial cells of the vas after treatment with the nuclear beta-Gal gene construction and of 13.3% after employing the GFP gene construction. Expression of the GFP gene appeared from one week up to three months following injection, and it appeared as patches of modified cells along the epithelium. Results from immunocytochemistry and Western Blotting support the conclusion that transfection of epithelial cells was achieved. We have also transfected the vas using gene constructions that express secretory proteins--specifically, the reporter system pSEAP-control that expresses a secretory form of human placental alkaline phosphatase, and the pGFP-Ctk-37 that expresses a secretion form of GFP. In both cases, the fluids expressed from the transfected vas showed a significant increase of alkaline phosphatase activity after pSEAP transfection and the presence of GFP protein when pGFP-Ctk-37 gene construction was employed. Our results indicate that the vas can be transfected in vivo using liposomes as vectors of foreign genes and that the vas fluid contents can be modified.

Increase in apoptosis and of the stress protein HSP70 in the mouse epididymis produced by the antiandrogen flutamide / El antiandrógeno flutamida produce un aumento de la apoptosis y de la proteína de stress HSP70 en el epidídimo del ratón

The general aim of this paper was to characterize some changes induced by androgen receptors blockage in the epithelial cells of the mouse epididymis. The antiandrogen flutamide was injected (10 mg/Kg b.w.) to adult male mice which were sacrificed 24h. and 72h. after. Controls injected with the vehicle (corn oil) were sacrificed at the same intervals. Cryosections were made of the epididymides and examined by the TUNEL method for quantification of apoptosis and also using immunocytochemistry to visualize the expression of the stress protein HSP70. The highest indexes of apoptosis were observed in the caput epididymis after 72 h. and were of 7.40 cells/1000 in contrast to controls (0.21 cells/1000). HSP70 appeared particularly increased in the caput and cauda epididymis after 72 h. treatment. Results indicated that the blockage of androgen receptors induces apoptosis and a HSP70 expression in the principal epithelial cells of the mouse epididymis, and that these changes occur in a region-specific fashion.

Este trabajo estudia los cambios inducidos por el bloqueador de receptores de andrógeno flutamida en el epitelio del epidídimo del ratón. Varios machos adultos fueron inyectados con flutamida (1Omg/Kg.b.w.) y se sacrificaron a las 24 y 72horas. Otros machos, que sirvieron de controles fueron inyectados sólo con el vehículo empleado para las inyecciones (aceite de maíz) y se sacrificaron a intervalos similares. Los epidídimos tratados y controles fueron examinados mediante el método TÚNEL para cuantificar la apoptosis y mediante procedimientos inmunocitoquímicos para localizar la proteína de stress HSP70. El índice apoptótico más alto fue observado en la cabeza del epidídimo después de 72 horas de tratamiento. HSP70 se observó también a las 72 horas en la cabeza y en la cauda epididimaria. Los resultados indican que el bloqueo de los receptores de andrógenos induce apoptosis y expresión de HSP70 en las células principales del epitelio epididimario, y que estos cambios ocurren afectando a regiones específicas del epidídimo.

Effect of Flutamide in Mouse Spermatogenesis and on the Function of Seminal Vesicle and Prostate

RESUMEN: La espermatogénesis está regulada por el eje hipotálamo-hipófisis-gónada, y los andrógenos juegan un rol fundamental en sus últimas etapas. La administración del antiandrógeno flutamida interfiere con ella y con la función de órganos andrógeno dependientes (próstata (P) y vesícula seminal (VS)). En este estudio se inyectó flutamida (10 mg/Kg peso corporal) a 10 ratones y vehículo al control(n=6). Los ratones se sacrificaron a las 24(n=5) y 72(n=5) horas. En cortes de testículo se midió el diámetro del túbulo (TD) y la altura del epitelio (EH). P y VS se maceraron, y se determinaron las concentraciones de fructosa (VS) y zinc (P). No existe diferencia significativa en el TD entre los grupos. Sin embargo, el grupo de 72 h presenta menor EH respecto al control (p<0.01). La fructosa es menor sólo a las 72 h (p<0.01), intervalo en el cual se presenta la mayor concentración de zinc (p<0.01). La disminución de la EH se explicaría por el desprendimiento de las espermátidas elongadas, debido al bloqueo del efecto de la testosterona, sin reflejarse en el TD a intervalo corto. La disminución de fructosa refleja claramente la deprivación de andrógenos, en tanto que la concentración prostática aumentada a 72 hrs sugiere deficiencia en la secreción de zinc.

Transfection of Gametes: A Method to Generate Transgenic Animals / Transfección de Gametos: Un Método para Generar Animales Transgénicos

The production of transgenic animals (TA) using transfected spermatozoa or eggs is commented. Different methods have been employed to introduce transgenes into the gametes of several vertebrates and invertebrates. Methods for the transfection of gametes have employed naked DNA, viral vectors, DNA/Liposome complexes, electroporation or high velocity microprojectiles. Spermatozoa and oocytes or eggs have showed a good transfection efficience (80% in some cases), and microscopical observations demonstrated that the transgenes appeared localized in the nucleus. Gametes have shown to be naturally protected against the entrance of foreign genes because some semino plasma or plasma membrane proteins block the entrance of foreign genes in spermatozoa. In the female this blockage is undertaken by the egg covers (the zona pellucida in mammals and the perivitelline coat in mollusks). In several cases the production of TA has been described after using the transfected gametes for in vitro fertilization or inseminations. Sometimes, larger percentages of TA were observed (85% in salmon). Nevertheless, these TA were mainly chimeric for the transgene and they were not capable to establish transgenic lines. It seems probable that TA produced by gamete transfections are different from those originated by the conventional microinjection procedures. Furthermore, gametes would develop some kind of mechanisms that modify the integration/expression of transgenes, or that block the integration of transgenes in the germinal line.

Se comentan algunos aspectos sobre la transfección de gametos y su empleo para la producción de animales transgénicos (AT). Para la transfección de gametos han sido empleados ADN desnudo, vectores virales, complejos ADN-Liposomas, electroporación, o microproyectiles de alta velocidad. En general, se han obtenido altos porcentajes de transfección (hasta del 80% en espermatozoides) y se ha observado que los transgenes se localizan en el interior del núcleo. Se ha constatado que los gametos están naturalmente protegidos frente a la entrada de genes extraños: en espermatozoides esta función la cumplen diversas proteínas presentes en los fluidos seminales o en su membrana plasmática; mientras que en los gametos femeninos son las envolturas del huevo (zona pelúcida en mamíferos o membrana perivitelina en moluscos) las que desarrollan esta labor. En muchos casos, los gametos transfectados se han utilizado en fecundaciones in vitro o en inseminaciones a fin de crear AT. En algunos casos, los porcentajes de estos AT han sido altos, como en el salmón (85%). Pero los AT, así creados, han sido en su mayoría quimeras y no han sido capaces de producir líneas transgénicas. Se sugiere que los AT producidos por gametos transfectados, son diferentes a los producidos por el método convencional de microinyección. Es probable que los gametos posean mecanismos, aún no descritos, capaces de modificar la integración/expresión de los transgenes, o que impedirían la integración de los transgenes en la línea germinal.

Ageing induces apoptosis and increases HSP70 stress protein in the epididymis of Octodon degus

Apoptosis has been largely analyzed in the testis. Nevertheless, the epididymis has been scarcely studied. We analyzed the number of apoptotic cells in the different regions (caput, corpus and cauda) of the epididymis of the South American rodent Octodon degus both young and senile. Apoptosis was identified using the TUNEL method which detects in situ DNA fragmentation. Apoptosis was detected in the principal cells of the epididymal epithelium. The caput epididymis was the region more affected. The caput of young animals showed that 0.32/1000 cells were apoptotic in contrast to 5.1/1000 of senile animals. Also in the cauda epididymis apoptosis is increased with age, appearing 0.14/1000 and 3.9/1000 in young and senile animals, respectively. On the other hand, we used a immunocytochemical method to localize the stress protein HSP70. HSP70 appeared notably increased in the principal cells of the cauda epididymis of senile animals. Changes in the epididymal epithelium are probably due to the low androgen levels existing in senile animals and are a region dependent phenomenon