Transgenic technologies for the study of epididymal function

R. John Lye, Barry T. Hinton

Department of Cell Biology, University of Virginia Health System, School of Medicine, Charlottesville, VA 22908, USA

Asian J Androl  2000 Mar; 2: 33-38

Keywords: epididymis; sperm maturation; transgenic organisms; fertility

Abstract

1 Definition of terms

In an ideal world, one would study the function of a protein by turning it off and then on again, at will, with both tight temporal and spatial control. While this is not yet possible, a recent paper from Utomo et al^[1] has taken a large step in this direction. We would like to put this paper into context by discussing what has already been done with respect to transgenic organisms in the investigation of epididymal function and show how these techniques might be applied to future studies of the epididymis.  

Classical genetics refers to the analysis of gene function by analysis of the phenotype resulting from either spontaneous or induced mutations. Following the recognition of the desired phenotype (for example, male sterility), one would perhaps map the affected gene to a chromosomal location, test for interactions (enhancement, rescue or epistasis) with other genes with similar phenotypes, or do biochemistry to identify the mutated protein. In any case, it would be important to clone the affected gene and identify the changes that result in the observed phenotype. Classical genetic studies are most easily done in organisms for which there is a good genetic map, in other words, in organisms that have become model systems such as Drosophila melanogaster (fruit fly), Caenorhabditis elegans (nematode worm), Saccharomyces cerevisiae (baker's yeast), Mus musculus (mouse), Chlamydomonas reinhardtii (flagellated protozoan) and Arabidopsis thaliana (wild mustard). Mutations that have multiple effects or times of action, especially lethal alleles, are rather difficult to study using classical techniques; however, a variety of clever tricks have been developed to get around this obstacle. One of these techniques is the use of conditional alleles that allow the gene to be active under one set of conditions (e.g. cold temperatures), while inactivating the gene under a complementary set of conditions (e.g. warm temperatures). Another technique for the study of such genes is the use of clonal chimeras. With this technique, a homozygous mutant allele is rescued with a chromosomal duplication which covers that region of the genome. This free duplication tends to be lost at some frequency during somatic cell growth since it will not pair properly at mitosis, resulting in an animal in which most cells contain the rescuing duplication, but which has patches of cells that have lost the duplication. These patches may be large if the duplication was lost early in development, and smaller if it was lost later on. Such techniques have allowed classical geneticists to gain considerable insight into the function of many genes.

Molecular genetics is used to refer to the suite of techniques that are available for manipulation of DNA sequences in vitro. These include, but are not limited to, techniques such as restriction enzyme digestion, DNA cloning, PCR mutagenesis, and site specific recombination. These techniques have become completely pervasive in modern biological studies, so much so that even classical genetic analyses generally utilize at least some molecular genetic techniques.

2 Transgenic techniques

A variety of techniques have been employed to introduce exogenous DNA into different organisms. Surprisingly, one of the simplest methods, direct injection of naked DNA, has been demonstrated to work in some cases. Injection of plasmid DNA into the germline syncytia of the nematode C elegans produced heritable transformants when a selectable marker was included^[3]. Transposons are DNA elements which are able to move around the genome when their associated transposases are activated. Because of this property, pioneers in the field of transgenic technology quickly realized that exogenous DNA could be carried along for the ride, if it were cloned into the transposon. This technology has been exploited in Drosophila where the P elements, a particular class of transposon, have been developed as integrative vectors for transformation^[4]. However, transposon-based vectors have been less successful in other organisms. 

Among the first successful methods for mammalian transgenesis was pronuclear injection. In this technique, the double-stranded exogenous DNA containing, in its simplest form, a promoter and a structural gene (for example a reporter gene like -galactosidase or GFP) is injected into one of the large pronuclei of a fertilized mouse oocyte. The exogenous DNA is integrated into the host genome at random sites, usually as multiple copies arranged as a concatamer. Both the copy number and the integration site can have large effects on the expression level of the introduced construct, so it is important to generate several transgenic mouse lines. The exogenous DNA must integrate into the host genome prior to the first cell division, otherwise the resulting embryo will be chimeric, with only some cells containing the exogenous DNA. If the germline receives and transmits the exogenous DNA, however, even a chimeric animal can be used in subsequent studies. If the inserted DNA happens to integrate into an existing gene, it may have a phenotype separate from that of the introduced DNA. This effect is known as insertional mutagenesis; although this can be a useful technique in its own right, it may interfere with the analysis of the gene of interest. Phenotypes due to insertional mutagenesis may be distinguished from those due to the exogenous DNA by analysis of several transgenic lines.  Since the insertion site is random, a consistent phenotype is suggestive of it being due to the introduced DNA rather than an effect of sequences surrounding the insertion site.

More recently, embryonic stem cells (ES cells) have been utilized to generate transgenic mice. Exogenous DNA is introduced into the ES cells by micro-injection, cationic lipids or electroporation and transformants are then selected using a positive selection method such as drug resistance; these transformed cells are then injected into a blastocyst. The resulting chimeric embryo is placed into a surrogate mother and carried to term. The resulting progeny are analyzed for the presence of the exogenous DNA (using PCR or Southern analysis, for example), and then tested to see if they will transmit it to their offspring.  When the exogenous DNA is introduced into the ES cells, it may integrate randomly; however, if sufficient genomic flanking sequence has been incorporated into the construct, and especially if a selectable marker (neomycin resistance, for example) is included, it is possible to select for cells in which homologous recombination has occurred. In this case, the introduced DNA replaces the endogenous gene; this is the basis for the so-called knock-out technology. Similar methods can be used to generate knock-outs (if the introduced DNA disrupts the gene resulting in a null allele), dominant negatives (if the introduced DNA has been mutated so as to interfere with the endogenous gene or with its binding partners) and over-expression constructs.

One aspect that may complicate the analysis of transgene phenotypes is the temporal regulation of the gene under study. For example, if the gene is active in early development, and again in adult life (multiple times of action), disruption of the embryonic activity may prevent that gene from ever being expressed in the adult due to a loss or disruption of the required target tissue. This problem has led to the development of systems for the conditional activation or inactivation of genes. The design of such a system requires the use of a ligand which is benign, well tolerated by the host and does not have an endogenous receptor. Two popular systems based on such a design are the Tet-On/Tet-Off system developed by Gossen and Bujard^[5] and the ecdysone-inducible system developed by No et al^[6]. The Tet-On/Tet-Off system uses the tetracycline resistance system from bacteria comprising the tetracycline sensitive repressor protein fused to the VP16 (virion protein 16 of Herpes simplex virus) transactivator; this chimeric protein will bind to its cognate DNA binding site and activate gene expression in a tetracycline sensitive manner.  The ecdysone-inducible system uses a fusion of the insect molting hormone (ecdysone) receptor and the glucocorticoid receptor as a heterodimer with the retinoid X receptor to bind to a synthetic response element.  With either conditional gene expression system, a variety of issues must be addressed when attempting to interpret the resulting phenotype. Among these concerns are the half-life of the inducing agent (tetracycline/doxycycline or ecdysone) and the half-life of the protein or gene product being affected.  Significant perdurance of the protein product due to either protein or message stability may hinder the ability to perceive a phenotype caused by a conditional gene disruption within the time course of the biological response to the stimulus. Another complicating factor for the analysis of transgene phenotypes is that the phenotype may be indirect. If one is attempting to determine the effect of a given gene on sperm maturation in the epididymis, an expected phenotype might be male infertility. Male infertility might result from non-epididymal causes such as behavioral defects, alterations in accessory glands or structures, or poor growth and sexual immaturity due to metabolic defects. 

A recent development in transgenic technology takes advantage of the Cre recombinase from the bacteriophage P1. This enzyme catalyzes a precise recombination between two lox-P sites. If these two sites are arranged as a tandem duplication on either side of a stretch of DNA, that DNA will be efficiently excised upon recombination (see Figure 1). However, if one site is in the genome and one is on a plasmid,  the plasmid will be inserted into the genome. Lox-P sites can be inserted into genes using the technique of homologous recombination in ES cells, and if they are inserted into introns, for example, they are usually well tolerated by the resulting embryo and will generally have no phenotype of their own. When the resulting mice are bred to a line of mice carrying an active Cre recombinase, the intervening DNA will be excised.  Since selection of recombinant ES cells requires the use of selectable markers, flanking these markers with lox-P sites (floxing them) allows the marker to be removed. This may be useful if the selectable marker confers a phenotype to the resulting mice. 

Figure 1.  Cre recombinase catalyzes the precise recombination of two lox-P sites (black arrows). A. If the two sites are arranged as a tandem duplication flanking a stretch of DNA (open box), the action of the Cre recombinase (X) will be to excise that stretch of DNA. B. If one lox-P site is in the genome and the other is on a plasmid, Cre recombinase will act to insert the plasmid sequence into the genome.

3 Transgenics and epididymal function

There are a variety of reports of transgenic mice with male infertility as a phenotype. Some of these cases may be due to disruption of epididymal genes, while others may be indirect. Few of these transgenics were made specifically to study the epididymis, but they display defects in fertility.  A selected group of the transgenics which result in male infertility will be described, in order to illuminate some of the advantages and disadvantages of this technique.

Aromatases are enzymes involved in steroid metabolism. Two different aromatase deficient mouse lines^[8,9] with differing phenotypes have been generated, despite being targeted disruptions of the same gene,cyp19. In one study, the mice had defects in sexual behavior; while in the other, the transgenic mice displayed an impairment of spermatogenesis. Whether these different phenotypes are due to methodological differences, or to genetic background differences between the mouse strains used, is not clear. 

A targeted disruption of the retinoic acid receptor alpha 1^[10] caused the epithelia of the epididymis and the vas to undergo a squamous metaplasia resulting in male infertility due to blockage of the normal sperm passage through the ductal system. 

There are two estrogen receptors, alpha and beta, and both are expressed in the male; male mice whose estrogen receptor alpha are disrupted (ERKO mice) are infertile^[11]. Estrogen regulates the re-absorption of water that occurs in the epididymis, and this process is blocked in the ERKO mice resulting in sperm diluted by excess luminal fluid. In addition, it is likely that maturation factors secreted by the epididymal epithelia may also be diluted, further contributing to the infertility. The lack of water re-absorption eventually leads to dilation of the seminiferous tubules and to degeneration of the seminiferous epithelium, resulting in impaired spermatogenesis.

The proto-oncogene c-raf-1 and the related gene A-raf have been shown to be expressed in the mouse epididymis^[12]. The epididymal phenotype of the knockout is unclear because of lethality associated with the loss of raf activity in one genetic background (C57 Bl/6) but not in another strain (129/OLA).

Male mice heterozygous for a targeted disruption of the apolipoprotein B gene were infertile^[13]. Their sperm were incapable of fertilizing eggs in vivo or in vitro. However, the sperm were able to fertilize oocytes in vitro once the zona pellucida was removed.  Fertility was restored upon introduction of the genomic sequence for the wild type human apolipoprotein B sequence as a transgene, confirming that the phenotype was due to the disruption of the mouse apolipoprotein B gene.

Sequences from the mouse mammary tumor virus (MMTV) direct protein expression to various ductal secretory epithelia, especially those of the mammary gland, kidney and epididymis.  Insertional mutagenesis of the int-3 locus^[14], which is highly homologous to the Drosophila Notch genean intercellular signalling receptor, resulted in complete male infertility due to severe hyperplasia of the epididymal epithelia. c-erbB-2 is a human receptor tyrosine kinase and it is an epidermal growth factor (EGF) receptor family member^[15].  All the male mice which were transgenic for a constitutively active, transforming allele of c-erbB-2 under the control of the MMTV long terminal repeat (LTR) were completely sterile^[16]. Severe hyperplasia of the epithelia of the epididymis, vas deferens and seminal vesicles was observed, presumably accounting for the infertility. The rat homolog of c-erbB-2 is c-neu; expression of an activated version of c-neu under the control of a truncated MMTV promoter^[17] resulted in infertile male mice. Consistent with the c-erbB-2 results, the c-neu transgenic mice showed bilateral epididymal hypertrophy and extensive epididymal epithelial hyperplasia. Male mice transgenic for an N-ras/MMTV construct showed reduced fertility^[18] which varied in its time of onset. Their sperm were grossly deficient in motility, and many of the sperm had detached heads. These sperm were ineffective in an in vitro fertilization assay irrespective of whether the zona was removed or intact.

Bone morphogenetic proteins (Bmp) are intercellular signalling molecules with diverse roles during development. Bmp8a and Bmp8b are tightly linked genetically and have similar expression patterns. Targeted mutations of Bmp8b have been shown to cause germ cell degeneration. In contrast, Bmp8a knockouts result in the degeneration of epididymal epithelia^[19] in addition to germ cell defects. 

A knockout of the proto-oncogene c-ros also resulted in male infertility^[20]. c-ros is an orphan protein tyrosine kinase receptor expressed at high levels in the developing epididymis and other tissues, including kidney, heart, lung and testis^[21], in the rat.  c-ros expression disappears from most tissues during development; however, c-ros expression is maintained at low levels in the adult kidney and at high levels in the initial segment of the adult epididymis. In the c-ros knockout male mice, loss of c-ros activity results in the failure of the initial segment to develop and differentiate. This lack of a developed epididymal initial segment leads to immotile sperm, highlighting the importance of this region for proper sperm maturation. 

-Glutamyl transpeptidase (GGT) is an enzyme involved in the glutathione cycle and it is an important component of cysteine metabolism. GGT is highly expressed in secretory or absorptive epithelia, including the kidney, pancreas, small intestine and seminal vesicle^[22]. Mice with a targeted disruption of GGT^[23] showed a coat color defect and a severe growth deficiency which could be rescued by feeding the mice N-acetylcysteine. In addition, GGT-deficient mice exhibited hypoplastic testes, seminal vesicles and epididymides. Disruption of the purinoceptor P2X₁^[24] results in male infertility due to a defect in sperm transport.  Sperm from these mice were motile and were capable of fertilizing oocytes in vitro. The infertility was found to result from a decrease in smooth muscle contraction in the vas deferens resulting in a reduction in sperm number in the ejaculate. 

4 Acknowledgements

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Correspondence to: Barry T Hinton,Department of Cell Biology, University of Virginia Health System, School of Medicine, Charlottesville, VA 22908, USA.
Tel: +1-804-924 2174  Fax: +1-804-982 3912
E-mail: bth7c@virginia.edu
Received 2000-01-25     Accepted 2000-02-28