Several abnormalities of the reproductive system are known to be of genetic origin, but only some specific types of reproductive anomalies are really characterized as genetic anomalies. Some of these include cryptorchidism and testicular hypoplasia. There are some basic genetic concepts that must be recognized before particular genetic factors can be determined to lead to abnormalities in the structures and functions of the reproductive system.
BASIC GENETIC CONCEPTS
Chromosomes
The total number of chromosomes is constant in all normal individuals within a particular species, and when arranged in pairs the chromosomes are identical for all members of that species. The paired chromosomes in mammalian somatic cells are called autosomes. The sex chromosomes are not autosomes; they are paired in mammalian females (XX; homogametic sex) and unpaired in males (XY; heterogametic sex). The mammalian X chromosome is usually larger than the Y chromosome. In birds, however, the female is the heterogametic sex (ZW) and the male is homogametic (ZZ). Thus, unlike in mammals, the female bird determines the sex of the offspring.
The normal chromosome number for a somatic cell is called the diploid number (2n) and is constant in normal individuals within a species. However, the gamete has one-half the diploid number of chromosomes and this is known as the haploid number (n). When a male and a female gamete unite at fertilization, the diploid number is restored.
The complete set of chromosomes is called the karyotype. The karyotype can be displayed by photographing, cutting out and arranging the chromosomes systematically according to cytogenetic conventions. Chromosomes can be classified according to the length of the chromosome and the position of the centromere seen at the metaphase stage. The centromere is a heterochromatic region (non-staining), which tends to constrict the chromosome at a specific position (primary constriction). If the centromere is situated halfway between the chromosome arms it is known as metacentric, and when located towards one end of the chromosome it is called submetacentric. However, if the centromere is located at the very end, the chromosome is referred to as acrocentric (Figure 13.1).
Figure 13.1 Types of chromosomes based on centromeric position: (a) metacentric; (b) submetacentric; (c) acrocentric; (d) telocentric; (e) metacentric with satellite arms. Metacentric and submetacentric are so-called biarmed chromosomes
Figure 13.2 Mechanisms resulting in triploidy by (a) polyandry and (b) polygyny
Figures 13.2 and 13.3 show different mechanisms resulting in triploidy by polyandry and polygyny, and mechanisms of non-disjunction and anaphase chromosome lag leading to aneuploidy. Figure 13.4 illustrates a map of imprinted regions in the human genome.
Genes and patterns of inheritance
Genes, located in a fixed position (locus) on a chromosome, are the basic units of inheritance. Each gene governs a specific trait (phenotype) and is recognized by alteration (mutation) of that particular gene which changes the phenotype of an individual who carries the mutant gene. Genes which control the alternative expression for specific traits are called alleles. Thus, the genotype of an animal is its complete set of genes, and the phenotype is the expression of these genes, whether as physical, biochemical or physiological traits.
A trait determined by a gene can be inherited either as an autosomal or sex-linked trait, and can also be either dominant or recessive. Traits that are governed by dominant genes express themselves when these genes are present on both chromosomes of a homologous pair (homozygous condition), or on only one of the chromosome pair (heterozygous condition). On the other hand, a recessive trait may only be expressed when both parents have contributed the mutant genes.
Figure 13.3 Diagrammatic illustration of the mechanisms of non-disjunction and anaphase chromosome lag leading to aneuploidy
Figure 13.4 Map of imprinted regions in the human genome, based on phenotypes detected in cases of non-apparent disomy involving the maternally inherited homolog (left chromosome in each pair) or the paternally inherited homolog (right chromosome in each pair). UPD, uniparental disomy. Revised from Ledbetter and Engel (1995)
Autosomal dominant traits affect both males and females. They tend to vary considerably in their severity (expressivity), and rarely skip a generation (non-penetrance).
Abnormalities of both structure and function of the reproductive system may be genetically determined. As most anomalies are the result of an interaction between environment and genotype, it is difficult to classify them strictly into inherited or acquired disorders. Most inherited abnormalities follow simple Mendelian inheritance, a few are acquired as sex-linked, and others as polygenic traits.
CHROMOSOME ANOMALIES
(FIGURES 13.5-13.7)
Chromosomal arrangements occur during mitosis and meiosis. Normally the ovum (X) is fertilized by sperm X or sperm Y. In abnormal cases of non-disjunction of oogenesis or spermatogenesis, fertilization may occur between an abnormal sperm and/or an abnormal egg, resulting in various chromosomal anomalies. Structural anomalies of the chromosomes include translocation, deletions, rings and inversions of chromosomes during either mitosis or meiosis (Figure 13.5). Such anomalies affect individual autosomes or sex chromosomes. The animal cell comprises a nucleus, protein-manufacturing units and energy-production points. Spiraling double strands of atoms are the DNA, the master chemical of genes. The sequence or layout of these atoms contains all the instructions the cell needs to function.
Aberration of chromosome number
Aberrations of chromosome structure and number are important because of their association with reproductive problems such as birth defects and embryonic or fetal death. Changes in chromosomes may be alteration in the total number of chromosomes, involving either addition to or loss of one or more chromosomes or set of chromosomes. Changes may also be structural, associated with rearrangement of chromosome segments within a chromosome or between two chromosomes. Modification of the chromosome number may affect a whole set of chromosomes such that, instead of the normal diploid (2n) number of chromosomes, triploid (3n), tetraploid (4n) or even pentaploid (5n) animals may result. This condition is known as polyploidy.
There are various mechanisms leading to these numerical changes. Mechanisms resulting in polyploidy include errors during meiosis (failure in the reduction of chromosome number) or at the time of fertilization (polyandry or polygyny), and abnormalities of the mitotic spindles at critical stages during fetal development.
Aberration of chromosome structure
A change in the structure of a chromosome is a result of one or more segmental breaks on that chromosome, with or without reunion of the broken segments. These can be divided into four major categories: deletion, duplication, inversion and translocation. In addition to these, misdivision of the centromere may occur occasionally, leading to the formation of an isochromosome (Table 13.1).
Deletion
Chromosomal deletion is a consequence of one or two breaks and the loss of a chromosome segment, and may be either terminal or intercalary. A terminal deletion occurs when there is a break and the loss of the terminal segment, or two breaks and the reunion of the broken ends to form a ring. On the other hand, intercalary (or interstitial) deletions involve the loss of a chromosome segment between two breaks. A chromosome segment devoid of a centromere (acentric fragment) is lost during cell division because it is unable to orient itself on the mitotic spindle. Deletions involving large chromosome segments are not seen in animals, because cells from which the chromosome segments are deleted are not viable. Nevertheless, if the segment deleted is small or genetically inactive (heterochromatic), the cell with the deletion may be maintained. For instance, a cell containing a deleted chromosome may be viable and capable of reproduction. However, its phenotype (or tissues containing cells with deletions) may be adversely affected. In a majority of cases, deletions can be identified using banding techniques, which enable segmental delineation of the chromosome.
Y chromosome microdeletion The prevalence of Y microdeletions is about 7% with a range of 1-35% depending on the patients selected for study and study techniques. Most Y chromosome microdeletions occur on the long arm (q) and are subdivided into three azoospermic factor (AZF) regions: a, b and c (Figure 13.6). Genes encoded in the AZF regions may be important in spermatogenesis. However, a small portion (up to 2%) of the fertile male population may also harbor very small microdeletions of the Y chromosome, probably involving non-coding regions. Before the advent of in vitro fertilization (IVF) with intracytoplasmic sperm injection (ICSI) and sperm retrieval techniques, such as testicular sperm extraction (TESE), many men affected by Y chromosome microdeletions could not reproduce. Choi et al. (2004) reported successful pregnancies with the use of sperm from Y microdeleted men and ICSI.
Figure 13.5 Diagrammatic illustrations of various structural aberrations: deletion, inversion, duplication and isochromosome
Table 13.1 Basic types of chromosomal alterations
Duplication
Duplications involve addition of an extra segment to a chromosome, through abnormal replication or unequal crossing over in meiotic cells of the parent. A duplicated segment can be terminal or intercalary, and at times shifted to another region. However, duplications are more common and less harmful than deletions. Instead, duplications of genes and segments of chromosomes are considered to be more important in evolution as they are believed to provide potential material for new gene combinations.
Inversion
Inversion involves two breaks on a chromosome and the rotation of the broken segment prior to reunion of the broken ends, resulting in the reversal of the gene sequence. Inversions can be either pericentric or paracentric. In pericentric inversion, the centromere is included in the inverted segment; however, for a paracentric inversion the centromere is not involved and it is confined to only one arm of the chromosome. Paracentric and pericentric inversions interfere with gametogenesis.
(1) Isochromosomes represent misdivision of the centromere and are always metacentric with identical but reversed gene sequence. For example, the metacentric bovine X chromosome when misdivided would give rise to a long and a short chromosome, each with two identical arms, that would actually be two chromatids.
(2) Translocation occurs when a portion of one chromosome is attached to a non-homologous chromosome. It often involves a reciprocal exchange of a segment between chromosomes. Reciprocal translocation is a result of an exchange between a segment of one chromosome and a segment of another. Translocation is relatively rare in humans and domestic animals.
(3) Interchange between segments of different chromosomes involving small segments of autosomes will be overlooked in farm animals such as cattle and goats, since the autosomes are all acrocentric in these species. Thus, the identification of translocated chromosomes is only possible with the aid of techniques such as banding.
In non-disjunction, both chromosomes move to the same pole, resulting in both daughter cells becoming aneuploid, since one of them will have a chromosome in excess of normal (2n +1) while the other will have one less than normal (2n- 1). In anaphase lag, one of the chromosomes falls behind and, thus, fails to be included into the nucleus of the daughter cells. This results in only one of the daughter cells being aneuploid.
Figure 13.6 Diagram of the Y chromosome depicting the location of azoospermic factor (AZF) regions a, b and c. SRY, sex-determining region of the Y chromosome. From Choi et al., 2004, with permission
In general, lacking a chromosome in the whole complement set is incompatible with life, while the presence of an extra chromosome disrupts the normal process of embryogenesis. When a large chromosome is associated in aneuploidy, the conceptus dies at an early embryonic stage of fetal development, regardless of the type of aneuploidy involved. However, an exception to this rule is X chromosome aneuploidy.
Abnormal spermiogenesis
A high incidence of abnormal sperm is linked with testicular hypoplasia. Testes of abnormal size, which exhibit inadequate growth and development, are considered to be hypoplastic. Although cryptorchid testes are hypoplastic, testicular hypoplasia also occurs with normal descended testes. Abnormal spermiogenesis may also cause sperm anomalies (Table 13.2).
X AND Y CHROMOSOME
BEARING SPERM
Figure 13.7 Diagrammatic illustrations of reciprocal translocation and Robertsonian translocation
Female animals have two similar sex chromosomes (X and X), whereas males have two different sex chromosomes (one X chromosome and one smaller Y chromosome). The gametes (egg and sperm) are haploid cells and contain either an X or a Y chromosome (Table 13.3). Diploid somatic cells of females (homogametic sex) contain a pair of X chromosomes, but somatic cells of males (heterogametic sex) have X and Y sex chromosomes. The genetic sex is determined in the oviduct at the time of fertilization, and the sex of the offspring is determined by the sex chromosome within the sperm.
Extensive investigations have been carried out for preselection and complete separation of X and Y sperm before artificial insemination. Sex of the offspring can also be predetermined in embryos arising from diploid or haploid nuclear transplantation into recipient ova, parthenogenetic activation of ova, or fusion of two oocytes. Different cytogenetic and cytological techniques are used to examine the diploid cells at an appropriate stage of fertilization to diagnose the genetic sex of the embryo. For example, fluorescence microscopy is used to detect the presence of a Y chromosome. Chromosome analysis is performed by culturing leukocytes or fetal cells to study the individual chromosomes using karyotyping procedures, which involve four concepts:
(1) Morphological, physiological, biophysical and immunological differences between X and Y sperm;
(2) Factors affecting the primary and secondary sex ratio;
(3) Techniques for separation of X and Y sperm based on valid statistical evaluation of the results;
(4) Attempts to alter the sex ratio by the use of ‘sexed’ sperm.
Molecular biology of sperm
When sperm are transported through the female reproductive tract, they undergo capacitation to acquire their fertilizing potential. Sperm release and/or acquire various micro- and macromolecules on their plasmalemma as they migrate through the vaginal secretions, cervical mucus, endometrial secretions, oviductal fluid and peritoneal fluid.
The degree of maturation and age of sperm in an ejaculate can influence density. Packed cell volume of bovine sperm is markedly affected by the osmolality of the medium. Live bull sperm placed in a hypo-osmotic saline solution swell to three times their normal size. Dead sperm do not swell or react osmotically. Live sperm placed in hyperosmotic media shrank from a volume of about 25 pm3 to 20 pm3.
Cytogenetics of X and Y sperm
There are many potential differences between sperm containing an X or a Y chromosome (Table 13.3). Sex chromosomes are responsible for any differences in DNA content. The presence of an X or a Y chromosome could cause a difference in the size, shape, weight, density, motility (type and velocity), surface charge, and surface biochemistry or internal biochemistry of sperm (Table 13.3). The degree of difference may also be affected by other factors such as the age of semen, repeat breeding (possible differential embryo mortality) and use of bulls that had been born as co-twins with heifers (and have circulating XX leukocytes).
There are species variations in the mass difference between X and Y chromosomes. The presence of large X chromosomes could result in greater weight and density of the X-containing sperm, if the size and other constituents are the same.
Table 13.2 Some inherited sperm defects affecting fertility
|
Defect |
Description |
Fertility |
|
Knobbed sperm |
Acentric thickening of acrosome |
Sterility |
|
Decapitated |
Head and tail separated at the neck region |
Sterility |
|
Dag defect |
Folding of tail over the middle piece of sperm,giving an impression of a swollen middle piece |
Infertility |
|
Pseudodroplet |
Rounded or elongated thickening of the middle piece |
Sterility |
|
Corkscrew |
Tail defect |
Infertility |
|
Diadem effect |
Nuclear pouch formation |
Infertility |
|
Sterilizing tail stump |
Tail defect |
Sterility |
Table 13.3 Some differences between X- and Y-bearing sperm
|
Parameter |
Difference |
Evaluation |
|
DNA size |
Less in Y sperm X sperm is larger |
Measurable and accepted Y sperm measured may or may not be representative of random sperm population |
|
Identify motility |
Y chromosome fluoresces Y sperm faster |
Species specific Evidence primarily dependent on accuracy of F-body staining technique |
|
Surface charge |
X sperm migrate to cathode |
No charge difference between X and Y sperm |
F, fluorescent
Sperm plasmalemma
The external plasmalemma has different characteristics in different parts of the sperm. X and Y sperm have different surface characteristics at spermiation from the germinal epithelium. If ejaculated, such differences would be masked by components that are absorbed from the seminal plasma.
Karyotyping of sperm
The distinction between X and Y sperm based on the fluorescence of the Y chromosome is facilitated by quinacrine staining of sperm, which causes fluorescence of the long arm of the Y chromosome, and a fluorescent spot (F body), which appears in 39-47% of sperm in smears stained with quinacrine. Banding techniques such as G-band and Q-band methods are recommended. Such procedures permit the accurate identification of individual chromosomes. Staining of chromosomes is carried out using a Giemsa dye mixture.
Technology to separate X and Y chromosome-bearing sperm
Extensive investigations have been conducted on X and Y chromosome-bearing sperm (Tables 13.3 and 13.4). Ten methods have been applied using: sedimentation of immobilized sperm in media; skimmilk powder, glycine, sodium citrate, glycerol; albumin column; velocity sedimentation; centrifugation through density gradients; motility and electrophoretic separation; isoelectric focusing; HY antigens; flow sorting by DNA content; and Sephadex column (Beernink, 1984; Bennet and Boyce, 1973; Bhattacharya et al., 1966; Corson et al., 1984; Ericsson and Glass, 1982; Hafs and Boyd, 1971; James, 1980; Meisstrich, 1982; Moore and Hibbitt, 1975; Pinkel and Gledhill, 1982; Sherbet et al., 1972).
Most of the techniques employed for sperm separation are based on non-equilibrium sedimentation (based on velocity of fall) or on equilibrium sedimentation on a density gradient (sedimentation to the level where specific gravities of sperm and the medium are equal). These techniques use simple gravity or centrifugation and are based on Stokes’ law of sedimentation of a rigid sphere through an incompressible, viscous fluid at a low Reynolds number (non-turbulent conditions). Aggregation of cells in the buffer is evaluated, and a concentration chosen at which aggregation is negligible.
Only two laboratory methods for separation of human X and Y sperm appear to be valid, reproducible and clinically applicable: albumin separation, which yields 75-80% Y sperm; and Sephadex filtration, which yields 70-75% X sperm. The separation of X and Y sperm is based on the following:
(1) Differences in the weight, density or size of the X and Y chromosomes as a result of differences in the size of different components of the sperm.
(2) Differences in haploid expression of X and Y chromosomes as a result of differences in the nature of sperm components: defective allocation (non-disjunction) of sex chromosomes, to either the gametes or, after fertilization, to early cleavage products, will result in individuals with somatic cells containing only a single X chromosome (XO; YO is lethal) or an extra X or Y chromosome (XXX, XXY or XYY) (Table 13.4).
Layered separation over albumin columns
When semen is layered over columns of serum albumin, increased numbers of Y sperm are recovered from the albumin layers. Semen aliquots of 0.5 ml (diluted 1 : 1 with Tyrode’s solution) are layered for 1 h over a 7.5% solution of serum albumin in a glass column (8 x 75 mm). The initial sperm layer is then removed by pipette, the albumin centrifuged at 2800-3200 rpm for 10 min, and the sperm disc is resuspended in Tyrode’s solution. The resuspended sperm is then layered over a two-layer serum albumin column. At 1 h, the sperm layer is removed, and after another 30 min, the 13% layer is removed. The 20% serum albumin is then centrifuged for 10 min, and the sperm disc is resuspended in 0.25 ml of Tyrode’s solution, which is then inseminated into the uterus.
Flow cytometry and sorting
With flow cytometry, high-speed measurements of components and properties of individual cells are made in a liquid suspension. Laser light is used for monochromatic illumination of cells stained with fluorescent dyes. Light detectors sample the fluorescence produced by the interaction of light with dye and produce electrical signals proportionate to the intensity of fluorescent light from each object. This technique is useful in the evaluation of the degree of separation needed to produce populations enriched in X or Y sperm.
Table 13.4 Techniques to separate X- and Y-chromosome bearing sperm
|
Technique |
Results |
|
Sedimentation of immobilized sperm on media Skim-milk powder, glycine, sodium citrate, glycerol Albumin column |
Insemination with sperm that had sedimented the greatest distance produced 70% females Increase in number of male offspring when sperm from the top layers was used Successful results with frozen sperm preselected on albumin column before cryopreservation |
|
Velocity sedimentation |
Sedimentation rates depend on size, density and shape of sperm Cell size difference is predominant factor in separation of types; shape is usually the least important factor Sperm heads have extremely aspherical shapes |
|
Centrifugation through density gradients |
Sperm separated according to their sedimentation rates by centrifugation through density gradients, provided the density of the gradient material is less than that of the sperm.The advantage is that the time required for separation is much shorter. Shorter time does not improve theoretical resolution of separation, because diffusion is insignificant |
|
Motility and electrophoretic separation |
Immotile sperm electrophoretically attracted to the anode at neutral pH.When electrophoretic separation is under conditions consistent with sperm motility, sperm migrate to the cathode. Sperm are oriented by electric field and swim in direction the head is facing. If negatively charged, sperm can be oriented so that the tail is facing the anode by virtue of its greater negative charge density and their intrinsic motility is greater than the electrophoretic mobility |
|
Isoelectric focusing |
Separation performed in columns with the fluid stabilized using density gradients Sperm layered on, or suspended in, this solution migrate electrophoretically until reaching an isoelectric point |
|
HY antigens |
Sperm treated with HY antisera. Insemination with mouse sperm treated with antisera to a Y-linked histocompatibility antigen produced 45% males compared with 53% for controls |
|
Flow sorting by DNA content |
Y-sorting is 72-80% successful. Disadvantages: low sorting rate and lack of sperm viability after sorting |
|
Sephadex column |
Some 70% of X sperm found in certain fractions of the filtrate when sperm were placed on top of a column of Sephadex 65-85% X sperm found in certain fractions of filtrate |
GENETIC ENGINEERING (FIGURES 13.8 AND 13.9)
Innovative technologies to enhance the efficiency of experimental gene transfer in different species are desperately needed. Such techniques would not only reduce the cost of individual projects, but would also increase the likelihood of new innovations in many disciplines. It is envisaged that procedures will be modified and enhanced as new advances are reported. There are several specific achievements that would significantly enhance experimental productivity:
(1) Development of alternative DNA delivery systems (e.g. liposome-mediated gene transfer or targeted somatic cell techniques);
(2) Identification of optimal conditions for a given gene transfer procedure and identification
of breeds best suited to the specific technologies;
(3) Complete animal genome mapping and identification of homologies to human genes;
(4) Establishment of routine and efficient germplasm culture and preservation systems (from preservation of gonadal tissue to culture and cryostorage of gametes).
Recent advances in genetic engineering have enabled scientists to uncover, rearrange and make copies, or clones, of genes. For example, each animal cell contains some 100 000 genes. At least 22 000 of these genes have been isolated, and some of their specific functions have been identified. Primary considerations for genetic manipulations and gene transfer are not only limited to management (including surgical manipulations and embryo handling), but also to expertise in tissue/cell culture, molecular biology techniques and sperm analysis/quantitation.
Figure 13.8 (a) Microinjection station includes a dissecting microscope (right) used for batch preparation of embryos and cells (and embryo transfer in mice) and microinjection microscope (left). Flanking the microinjection microscope, micromanipulators aid in precise manipulations with embryos, cells and buffers. (b) Pipette puller. Microneedles can be prepared with uniform tapers and shapes using an electronic or gravity-driven pipette puller. (c) Microforge. Needles may be fire-polished or bent to desired specifications using the microforge assembly that includes a microscope assembly for visualization of the finished product
Figure 13.9 Nuclear transfer and microinjection. (a) Nuclear transfer and cloning. Cells from blastocysts (e.g. inner cell mass cells) or other somatic tissues are obtained and grown in culture.These cells are used as nucleus donors for transfer into enucleated oocytes. In contrast to DNA microinjection, this genetic-engineering process includes an electrofusion step to fuse the transferred nuclei and enucleated oocytes. The fused ‘couplets’ (transferred nucleus plus oocyte) are transferred to recipients and live-born offspring are then evaluated for the genetic modification. (b) Microinjection of a zygote. Most zygotes are lipid-rich and relatively opaque
Table 13.5 Definitions, basic principles and types of human embryonic stem cells. Adapted from Amit et al., 2003 and Revelli et al.,2003
|
Cell type/principle |
Definition |
|
Embryonic stem (ES) cells |
Derived from the preimplantation embryo Pluripotent, capable of differentiating into representative cells of all three germ layers of the embryo Immortal, with long-term proliferation at the undifferentiated stage (self-maintenance) Maintain normal karyotype after prolonged culture Capable, after injection into blastocysts, of contributing to all three embryonic germ layers, including the germ line, and into mature chimeric animals Express unique markers, such as transcription factor Oct-4 or cell-surface markers like stage-specific embryonic antigen |
|
Embryoid bodies (EBs) |
Tumor-derived cell lines able to differentiate in vitro into a variety of cell types, including muscle and nerve cells, to aggregate into cell clusters in which part of the cells differentiate spontaneously, to form teratocarcinomas after their injection into recipient |
|
Cloning |
Development of a colony of cells from one cell by repeated mitosis: all cells have the same genetic constitution Transplantation of a somatic cell nucleus into an ovum for the purpose of developing an embryo through asexual reproduction |
Maternal-to-zygote transmission
Maternal-to-zygotic transmission has at least three functions that are required for continued development:
(1) Destruction of oocyte-specific transcripts, such as the RNA-binding protein MSY2 and histone H100, that are not subsequently expressed. The destruction of these mRNAs restricts the period of time during which the genes can function.
(2) Replacing of maternal transcripts that are also degraded during oocyte maturation and following fertilization, and that are common to the oocyte and the early embryo (e.g. actin) with zygotic transcripts. If these maternal transcripts are not replenished, development will shortly arrest owing to the embryo’s inability to execute basic cellular functions. Expression of these genes does not result in reprogramming of gene expression in the classical sense; nonetheless, their expression is essential.
(3) Promoting of the dramatic reprogramming in the pattern of gene expression that is coupled with the generation of novel transcripts not expressed in the oocyte. This reprogramming of gene expression is likely to be the molecular basis for the transformation of the differentiated oocyte into totipotent 2-cell-stage blastomeres.
Stem cells
The definitions, basic principles and types of human embryonic cells are summarized in Table 13.5. The body contains several self-renewing systems, including hematopoietic tissue, intestinal epithelium and epidermis. Spermatogenesis originates from spermatogo- nial stem cells, and the continuous production of progenitor cells supports male reproduction throughout adulthood. Extensive investigations were conducted on allergenic stem cell transplantation, the hematopoietic system and transplantation of allergenic hematopoietic stem cells for therapy of several hematological malignancies, metabolic disorders and solid tumors. Future research is needed to evaluate the immunological response to transplantation of other types of allergenic stem cells, particularly how they are affected by histoincompatibility. A technique was developed to transfer spermatogonial stem, in which the stem cells are transplanted into the seminiferous tubules of infertile recipient animals. The transplanted stem cells proliferate on the basement membrane and establish colonies of spermatogenesis. Spermatogonial stem cells are unique among the many types of stem cells in that they reside in an immunologically privileged environment.
Several methods have been used for gene transfer. The advantages and disadvantages of DNA microinjection and retrovirus-mediated gene transfer are described in Table 13.6.
Transgenic animal models have been applied in the fields of embryology, endocrinology, genetics, immunology, neurology, oncology, pathology, physiology, toxicology and virology (Table 13.7).
Table 13.6 Advantages and disadvantages of different methods of gene transfer. Adapted from Pinkert (2000) (personal communication)
|
Technique |
Advantages |
Disadvantages |
|
DNA micro-injection |
Relatively high frequency of generating transgenic animals (20-30% of live-born offspring) |
Random and potentially significant influence that the site of integration may exert on transgene expression (positional effects) |
|
High probability of germ-line transmission of the transgene Relative lack of constraints on the size or type of DNA construct used |
Potential for undesired insertional mutagenesis Occasional production of mosaic founders |
|
|
Relative stability of the transgene as it is transmitted from generation to generation |
Occasional lack of germ-line incorporation |
|
|
Low frequency of mosaicism or double integrations (combined estimate of 10-30% of founders) |
Time and expense required to obtain micromanipulation and microinjection skills |
|
|
Retrovirus-mediated gene transfer |
Uncertain |
Low copy number integration Additional steps required to produce retroviruses Limitations on the size of the foreign DNA insert (usually 9-15 kb) transferred Potential for undesired genetic recombination that may alter the retrovirus High frequency of mosaicism Possible interference by integrated retroviral sequences on transgene expression |
Genetic manipulation of embryonic stem cells
Genetic manipulation can be performed in human embryonic stem (ES) cells. Extensive investigations have been conducted on differentiation of human ES cells in both spontaneous- and directed-differentia- tion models. For example, human ES cells have been spontaneously differentiated into cardiomyocytes with their characteristic features: typical myofibril organization consistent with early stage cardiomyocytes, positive staining with specific cardiomyocytic markers including anticardiac myosin heavy chain, anti-a-actinin, antidesmin, expressed specific cardiac genes and physiological features of cardiac cells. At present there are more than 70 human ES cell lines in several laboratories around the world (National Institutes of Health, NIH; index.htm). Derivation of human ES cells can reproduced with reasonable success. The most common procedure used to isolate the inner cell mass (ICM) is immunosurgery, with the aim of selectively isolating the ICM of the blastocyst from the outer layer of the trophoectoderm. The zona pellucida is removed using Tyrode’s solution, and the embryo is exposed to antihuman whole antiserum.
Interspecies somatic cell nuclear transfer
Securing human blastocysts is a prerequisite factor for developing novel medical biotechnologies, such as cell replacement therapy using ES cells. However, an inevitable increase in the use of human embryos is anticipated with continuing technical improvements and their subsequent clinical application. Recent development of somatic cell cloning makes it possible to obtain human blastocysts without the use of human embryos; however, this raises concerns about human cloning and provokes ethical concerns. To date, strict regulation has prohibited human cloning and several countries have even limited the production of human cloned embryos.
To obtain human blastocysts, an interspecies somatic cell nuclear transfer (iSCNT) technique using human somatic cells and animal enucleated oocytes was suggested. Although disputes and serious concerns about interspecies cloning have been provoked, iSCNT is the only alternative to avoid the abuse of human embryos and even oocytes. The feasibility of interspecies cloning has been demonstrated in previous studies; blastocyst formation was possible after the transfer of mouse, bovine, porcine or rabbit somatic cells into bovine enucleated oocytes. It has now been applied to various animal research models. In terms of accessibility and ethics, human cord fibroblasts and enucleated bovine oocytes were selected as somatic cell donors and recipient cytoplasts.
DNA microinjection
Several experimental studies have been conducted on DNA microinjection, egg cytoplasm/gene control in development, translation of globin mRNA transgenic technology and altering the genome by homologous recombination. However, all these experiments were conducted on farm animals, using the mouse model (Brinster et al., 1980; Cappechi, 1989; Gurdon, 1977;
Hogan et al., 1994; Houdebine, 1997; Monastersky and Robi, 1995; Pinkert, 1994a; Pinkert, 1994b; Pinkert, et al., 1997; Pinkert and Murray, 1998). Very little is known about the possible application to human reproduction.
DNA microinjection techniques
DNA microinjection generally involves the use of micromanipulators and air- or oil-driven microinjection apparatus physically to inject the DNA construct solution into the ova. Almost any cloned DNA construct can be used. With few exceptions, microinjected gene constructs integrate randomly throughout the host’s genome, but usually only at a single chromosomal location (the integration site). This fact can be exploited to co-inject simultaneously more than one DNA construct into a zygote. The constructs will then co-integrate together at a single, randomly located, integration site. During integration, a single copy of multiple copies of a transgene (actually as many as a few hundred copies of the particular sequence) are incorporated into the genomic DNA, predominantly as a number of copies in head-to-tail concatemers. Because DNA microinjection is usually accomplished in ova at the one-cell stage, transgene incorporation occurs in essentially every cell that contributes to the developing embryo. Incorporation of the transgene into cells that will eventually contribute to development of germ cells (sperm or ova) is a common occurrence with this method.
Host DNA near the site of integration frequently undergoes various forms of sequence duplication, deletion or rearrangement as a result of transgene incorporation.
Advantages of the DNA microinjection method are:
(1) Relatively high frequency of generating transgenic animals (20-30% of liveborn offspring);
(2) High probability of germ-line transmission of the transgene;
(3) Relative lack of constraints on the size or type of DNA construct used;
(4) Relative stability of the transgene as it is transmitted from generation to generation;
(5) Low frequency of mosaicism or double integrations (combined estimate of 10-30% of founders) (Rosnina et al., 2000).
In contrast, disadvantages of this method include:
(1) Random and potentially significant influence that the site of integration may exert on transgene expression (positional effects);
(2) Potential for undesired insertional mutagenesis;
(3) Occasional production of mosaic founders;
(4) Occasional lack of germ-line incorporation;
(5) Time and expense required to obtain micromanipulation and microinjection skills.
Transgene-encoded mRNA expression
While absence of mRNA expression is probable for loss-of-function models and may only represent a confirmatory step of minor significance, the analysis of expression of the transgene is absolutely essential in determining the usefulness of a particular transgenic animal. Technically, the most critical step in analyzing transgene expression is the isolation of RNA. Care must be taken to avoid contamination of RNA preparations with ribonucleases (enzymes that degrade RNA). The presence of a specific mRNA is usually determined by RNA slot blot or Northern blot hybridization. Northern blotting is more informative as it confirms not only the presence but also the size of the mRNA transcript of interest (Rosnina et al., 2000).
Transgene-encoded protein expression
Various techniques are often employed to identify the unique protein or perhaps a specific enzyme activity. The immunoblotting technique, in which proteins are resolved on a polyacrylamide gel, transferred to a membrane and detected with a labeled antibody, is useful in verifying the appropriate molecular weight of the protein product of interest. To identify which cells within a tissue contain the protein product of the transgene, immunohistochemical staining of tissue sections with a labeled antibody can also be employed. Additionally, the use of ‘reporter genes’ (e.g. an oncogene, or a growth hormone or fluorescent protein gene) can often simplify determination of expression levels by producing a protein that is easily and unequivocally determined.
CLINICAL PARAMETERS
Preimplantation genetic diagnosis
Preimplantation genetic diagnosis (PGD) involves the assessment of the chromosomal status of an embryo in vitro, before its transfer to the uterus. Biopsy of one or two blastomeres is performed on day 3 of development, when the embryo is at the 6- to 10-cell stage. Each isolated cell is fixed and screened for chromosomal content using fluorescence in situ hybridization (FISH) with chromosomespecific DNA probes. PGD is applied to determine the sex of embryos from patients with X-linked disease and to identify chromosomally abnormal embryos from patients with an altered karyotype. Although blastomere culture may increase the number of cells available for chromosomal analysis, the high frequency of nuclear defects and the occurrence of polyploidy and mosaicism among cultured cells discourage the use of blastomere isolation and proliferation strategy for use in preimplantation genetic diagnosis.
Extensive investigations are needed in the areas of male sex determination, prenatal differentiation, postnatal development, cellular organization, paracrine regulation, male germ apoptosis, gonadotropin receptors, septo-optic dysphasia, related malformations, magnetic resonance imaging of hypothalamic- pituitary region in non-tumoral hypopituitarism; clinical aspects of interest include cancer of the male reproductive organs, blood-testis barrier, testicular descent and the prevention of male infertility after gonadotoxic cancer treatment in prepubertal boys.
Such information will be valuable for scientists, general pediatricians, endocrinologists and andrologists.
Identical twins
Identical twins have identical genes, as do a clone and its progenitor. Identical twins do look alike and even teachers mix them up. Identical twins are strikingly similar in many ways, but research shows that they are not completely alike. Identical twins are more identical than clones will ever be. As a group, identical twins are more similar to each other in personality than ordinary siblings or fraternal twins, who develop from two separate eggs; this is true even when the identical twins are apart.
Even identical twins are influenced by non- genetic factors, beginning in utero and extending to parents, friends, opportunities in life and chance occurrences. Since a clone and its progenitor would be born into different families at different times, these non-genetic factors could be expected to be more powerful. In the case of identical twins, there are also some genetic differences that are surprising.
Schizophrenia is clearly influenced by genes, for example. However, if one identical twin has schizophrenia, the chance that the other also has it is only 40-50%. Not even physical traits like height and weight are perfectly duplicated between pairs of identical twins. Height is among the traits most heavily influenced by genes, but identical twins may differ by as much as 4 inches. The correlation between identical twins is strong for height, strong for IQ, weaker for weight and weaker still for personality. Sexual and religious attitudes exhibit the same level of genetic influence as does personality, while attitudes on taxes and politics appear to be less influenced by genes. These findings are only population averages and cannot be used to predict anything about similarities between individual clones and their progeny. The degree of similarity for various traits roughly follows this pattern in identical twins. There is no perfect replica.
Cloned food/federal agencies
According to the FDA, 400-500 cows and bulls have been produced by cloning. The FDA’s Veterinary Medicine Advisory Committee stated that there are no biological reasons, based on either underlying scientific assumptions or empirical studies, to indicate that consumption of edible products from clones of cattle, pigs, sheep or goats poses a greater risk than consumption of those products from their non-clone counterparts. The National Academy of Sciences declared that while food from such clones posed a ‘low level of food safety concern’, more data were needed.
FUTURE RESEARCH
Additional research is needed in the following areas:
(1) Sperm motility: sperm isolate themselves based on different progressive motility.
(2) Sperm dimensions or density: cells sorted through a device sensitive enough to detect minute differences.
(3) Sperm cytogenetics: chemical or immunological reaction capable of selecting on sex chromosome content.
(4) Sperm environment: hormone or chemical condition resulting in penetration of the egg by an X or Y sperm.
(5) Search for Robertsonian chromosome rearrangements that include a sex chromosome: the availability of such chromosomes could facilitate specific breeding schemes suitable for sex selection.
(6) Cytogenetic sexing of cells isolated from an embryo and transfer of the selected embryo after short in vitro culture.