Extensive investigations have been conducted on the genes expressed in various reproductive processes (Abdel-Rahman et al., 1995; Agulnik et al., 1994; Ariel et al., 1994; Bathgate et al., 1996; Branford et al., 1997; Cao et al, 1995; Check et al., 1996; Cornwall and Hann, 1995; Domashenko et al., 1997; Furuya et al., 1995; Gerwin et al., 1995; Juneja and Koide, 2000; Nayernia et al., 1996; Sarge et al., 1994; Vani and Rao, 1997; Worrad and Schultz, 1997; Xu et al., 1995). Many genes coding for important factors and receptors are expressed during the development of the testes (Figures 14.1 and 14.2; Table 14.1). The relaxin-like factor (RLF) gene is highly expressed in the testes.
Figure 14.1 Schematic representation of the generation of two different progenies by fertilization between two distinct populations of sperm and eggs
MOLECULAR PARAMETERS OF CHROMOSOMES/GENES
Deoxyribonucleic acid
Deoxyribonucleic acid (DNA) is the molecule that contains and transfers genetic information from one generation to the next. DNA molecules, in association with histones and various proteins, are organized to form complex intracellular structures called ‘chromosomes’. Mammalian gametes are the direct products of the reductive division of chromosomes, termed meiosis, and thus contain the haploid number (abbreviated n) of hereditary determinants. Both gametes contribute equally to the genetic make-up of new progeny by transmitting the haploid number (n) of chromosomes to the newly formed diploid zygote (2n) of the next generation. The general mechanisms and genes expressed during spermatogenesis, fertilization and implantation are shown in Table 14.1. DNA contains hereditary information within its helical structure, and also provides the basis for the evolutionary process.
Figure 14.2 Series of events occurring during spermatogenesis
Table 14.1 Genetic control of testes determination
Gene |
Role during testes development |
Tdy (testes determining factor in mice) |
Act on supporting cell lineage and inducing differentiation of supporting cells to Sertoli cells |
Sry (Y-chromosome specific gene) |
Different homologs of Sry have a common open reading frame which has 41% homology to a DNA-binding motif HMG-box; Sry-encoded protein might have DNA binding activity |
Sox6 (Sry-related gene), Sox5 |
Overlapping functions in the regulation of gene expression during spermatogenesis in the adult mouse |
Sox9 |
Critical Sertoli cell differentiation factor |
TAZ83 |
Coding at early to midpachytene germ cell stage |
TAZ4 |
Testes specific gene located on chromosome 11 |
TNZ1 |
Expressed in neonatal Leydig cells |
DNA directs cellular growth and proliferation, and also regulates the differentiation of the fertilized egg to form the multitude of specialized cells required for organogenesis and proper functioning of mature animals. The majority of DNA is located within the nucleus in the form of coiled rods and is an integral part of chromosomes. Each species of farm animal has a fixed number of chromosomes, and each chromosome occurs as duplicates (homologous chromosomes). During the formation of gametes, reductive division, designated as meiosis, occurs, whereby the number of chromosomes in the cells is reduced to a haploid number (n), or half the diploid number (2n), found in somatic or non-sex cells. Men are the heterogametic sex, since one pair of sex chromosomes consists of an X and a Y type. This produces two distinct populations of spermatozoa, one bearing an X and the other a Y chromosome (Figure 14.1). The male gametes determine the sex of the new progeny. Following advances in genetics and techniques for separating chromosomes, X- or Y-bearing sperm can be selected and used to fertilize an egg and obtain progeny having the desired sex. However, there are no known inherent advantages of fertilizability between X- and Y-bearing gametes.
CELLULAR PROTEINS
Tyrosine phosphorylation plays an important role in cell growth, differentiation and apoptosis. Src family tyrosine kinases (SFKs) are key signaling molecules regulating the tyrosine phosphorylation of a number of cellular proteins.
Recombinant proteins
Synthetic human proteins are useful therapeutic agents. Polypeptides are simple to make: a gene is transcribed, the message translated, and the resulting protein may not even require post-translational modification. In other words, little intermediary metabolism is needed to produce most proteins; such products can be secreted directly into the milk of transgenic animals. Advantages of recombinant protein production over purification from source (e.g. from animals or human cadavers) include greater consistency of product quality, higher efficiency of production, lower immunogenicity than animal proteins, reduced infectivity compared to tissue-derived products, and genetic engineering of ‘super proteins’ with high activity. The only disadvantage of recombinant technology is that such proteins are difficult to detect when used for illicit purposes - the abuse of erythropoietin and growth hormone by athletes (Tseng, 1980).
MOLECULAR PARAMETERS OF SPERMATOGENESIS
Extensive investigations have been conducted on the genetic control of testes determination, genes involved in testicular development and function, Y-linked genes, spermatogenesis, T-type Ca2+ channels and alpha1E expression in spermatogenic cells, and their possible relevance to the sperm acrosome reaction (Chandley and Cooke, 1994; Harley, 1993; Hutchinson, 1993; Johnson and Barry, 1995; Lamb, 1995; Lievano et al., 1996).
The two compartments of the testes, seminiferous tubules and interstitium, are under the hormonal control of gonadotropins, acting on either Sertoli cells or Leydig cells. During spermatogenesis, these different cell types, and in particular germ cells, express four specific sets of genes, which are regulated at either the transcriptional or transductional level. Some are detected exclusively in haploid cells, whereas others are initially expressed before or during meiosis and continue to be expressed in spermatids:
(1) Male germ cell-specific gene homologs (e.g. glyceraldehyde-3-phosphate dehydrogenase);
(2) Unique genes expressed exclusively during spermatogenesis;
(3) Germ cell-specific alternate transcripts leading to specific isoforms;
(4) Genes developmentally regulated during germ cell differentiation (e.g. c-Abl protooncogene).
Mitosis
Germ cells present in immature testes are reactivated at puberty and undergo mitosis, while situated in the basal compartment of the tubule. These generated spermatogonial stem cells give rise to cells with distinct morphological characteristics, classified as A1 spermatogonia. The emergence of these cells marks the beginning of spermatogenesis. Each spermatogonium undergoes a defined number of mitotic divisions unique to the species, giving rise to a ‘clone’ of cells which differ morphologically from their parents. The nuclear division during mitosis (karyokinesis) is complete, whereas the cytoplasmic division (cytokinesis)
is incomplete, resulting in primary spermatocytes connected by thin cytoplasmic bridges. This interconnecting syncytial state of germ cells exists throughout the entire process of spermatogenesis.
Meiosis
During meiosis, a number of genes present on autosomes are expressed; however, genes located on sex-chromosomes remain inactive. Later during spermatid formation, the autosomes cease RNA production and their DNA becomes highly condensed or heterochromatic.
Postmeiotic phases
Spermiogenesis involves major remodeling of the structure of spermatids to form spermatozoa, which are composed of three distinct parts: head, middle piece and tail. The nucleus, which is present in the head region, contains compact packaged haploid chromosomes. On completion of spermatogenesis, the spermatozoa are released into the lumen of the seminiferous tubules by spermiation. Different species have varying periodicity in the spermatogenesis cycle. Nonetheless, within a given species the rate of progression of germ cell differentiation through the various stages of spermatogenesis is remarkably constant.
Spermatogenesis provides an interesting system for examining the regulation of gene expression during development and differentiation. The genes expressed during spermatogenesis can be divided into two main groups: diploid-expressed genes, e.g. the proacrosin gene, and haploid-expressed genes, e.g. the gene encoding the transition protein 2, a novel gene from the rat protamine gene cluster (Prm3), and a spermatid-located calpastatin gene. A number of novel genes that play a role during spermatogenesis have been identified (Tables 14.2 and 14.3).
MOLECULAR ASPECTS OF FERTILIZATION
Immotile and immature spermatozoa undergo a series of changes during their transit through the rete testes, ductuli efferentia and epididymis to become motile and mature. Expression of the BE-20 epididy- mal protein gene is detected in the cauda epididymis and the proximal segment of the ductus deferens, suggesting a role in sperm maturation and its capacity to fertilize ova.
Sperm-egg interactions
Sperm-egg binding between the outer membrane of the sperm and the outermost glycoprotein covering (zona pellucida 3, ZP3) of the egg occurs initially as a species-specific interaction. The sperm receptor present on the surface of the unfertilized egg involved in sperm-egg binding has been identified as ZP3. Sperm-egg binding triggers the acrosome reaction in the sperm head, which involves fusion of the outer acrosomal membrane with the overlying sperm plasma membrane. The net result of the acrosome reaction is the release of hydrolytic enzymes and exposure of the inner acrosomal membrane. Together with increased motility (hyperactivation), and the liberated hydrolytic enzymes, the acrosome-reacted sperm has the potential to penetrate its way through the zona pellucida and enter the perivitelline space. The sperm head next sinks into the oocyte, triggering the second meiotic division, which leads to the formation and extrusion of the second polar body.
Table 14.2 Genes expressed during reproduction
Reproductive process |
Gene |
Spermatogenesis |
Proacrosin gene Smcy, Hya, Sdma, CRES Spx1,MSH2,HSF2 Pgk2,ApoA1,Oct3/4 |
Oogenesis |
RLF gene, oxytocin gene |
Fertilization |
BE-20 |
Preimplantation and |
Cytokines, Sry |
implantation |
Myc, Spl Oct4, Oct6, PIBF EGF |
MOLECULAR ASPECTS OF INFERTILITY
Translocation and preimplantation genetic diagnosis
Translocation carriers have a high risk of infertility, recurrent spontaneous abortion and conception of a chromosomally abnormal pregnancy. Unbalanced embryos are as likely as normal embryos to develop to the blastocyst stage, thus implanting and resulting in spontaneous abortion and/or affected offspring at the same rate (Escudero et al., 2003). Preimplantation genetic diagnosis (PGD) is offered to carriers of translocations as an alternative to prenatal diagnosis and pregnancy termination of unbalanced fetuses. PGD for translocation is performed using several techniques of fluorescence in situ hybridization (FISH). Patients undergoing PGD of translocations obtain a pregnancy rate comparable with routine in vitro fertilization (IVF) patients and a very significant reduction in the frequency of spontaneous abortions. Patients with < 65% chromosomally abnormal sperm have a good chance of conceiving; patients with higher rates would need to produce ten or more good-quality embryos to have a reasonable chance of conceiving (Escudero et al., 2003).
Table 14.3 Genes involved in spermatogenesis and their function
Gene |
Role during spermatogenesis |
Smcy |
Y-chromosome gene; isolated from the region encoding spermatogenesis gene Spy |
Hya, Sdma |
Regulatory role in the expression of the male-specific minor histocompatibility antigen HY |
CRES (cystatin-related epididymal and spermatogenic) gene |
Specialized role during sperm development and maturation |
YRRM (Y-located RNA recognition motif) gene Spxl (X-linked homeobox gene), MSH2 (mismatch repair gene), HSF2 (heat shock factor 2) |
Candidate for azoospermia factor Regulatory role during spermatogenesis |
Pgk2,ApoA1,Oct3/4 Gene encoding T-type Ca2+ channel |
Role in sperm maturation process Voltage-sensitive calcium-channel sensitive to blocking agents dihydropyridine and nifedipine |
Growth factors
Interleukin-10 (IL-10) is an apheliotropic cytokine secreted by leukocytes and somatic cells with well characterized anti-inflammatory and immune-deviating properties. IL-10 inhibits proliferation and cytokine synthesis in type-1 T lymphocytes, and does not induce responsiveness or energy. Conversely, IL-10 favors differentiation and function of lymphocyte subsets with pivotal roles in immune tolerance, namely the newly discovered T regulatory (Tr1) cells and T helper cells. The ability of IL-10 to skew immune outcomes is indirect and is mediated through programming antigenpresenting cells preferentially to support expansion of alternative types of T cells. Moreover, IL-10 terminates inflammatory responses and limits inflammation- induced tissue pathology by inhibiting synthesis of tumor necrosis factor-а (TNF-a), interleukin-1, and a large array of other proinflammatory cytokines and chemokines in monocytes and macrophages.
Cell transfection
‘Stable transfectants’ are cells that permanently express a recombinant DNA sequence, or construct cells. In these cells the target DNA is stably integrated into the genome. Genomic integration may be achieved using a selectable marker linked to the gene of interest. Such integration may take place during genetic recombination; DNA modified by such incorporation is known as ‘recombinant’. Alternatively, cells may express the inserted gene for only a few days (typically 12-72 h) before ejecting the plasmid DNA. These transient transfectants are used for short-term experiments that demand gene expression without accurate simulation. Gene expression can be observed by intracellular microinjection of specific mRNA transcripts - either ‘sense’ or ‘anti-sense’. This technique is limited to short-term experimental analysis of small cell numbers.