Chapter 3 – Gametes and Gametogenesis




Abstract




After a blastocyst has implanted in the uterus and begins to differentiate into the three primary germ layers, a special population of cells develops as primordial germ cells (PGCs). These are destined to become the gametes of the new individual: future reproduction of the organism is absolutely dependent upon the correct development of these unique populations of cells. They originate immediately behind the primitive streak in the extraembryonic mesoderm of the yolk sac; toward the end of gastrulation they move into the embryo via the allantois, and temporarily settle in the mesoderm and endoderm of the primitive streak. In humans, PGCs can be identified at about 3 weeks of gestation, close to the yolk sac endoderm at the root of the allantois.





Chapter 3 Gametes and Gametogenesis




Gamete Precursor Cells: Primordial Germ Cells


After a blastocyst has implanted in the uterus and begins to differentiate into the three primary germ layers, a special population of cells develops as primordial germ cells (PGCs). These are destined to become the gametes of the new individual: future reproduction of the organism is absolutely dependent upon the correct development of these unique populations of cells. They originate immediately behind the primitive streak in the extraembryonic mesoderm of the yolk sac; toward the end of gastrulation they move into the embryo via the allantois, and temporarily settle in the mesoderm and endoderm of the primitive streak. In humans, PGCs can be identified at about 3 weeks of gestation, close to the yolk sac endoderm at the root of the allantois. These cells have many special properties in terms of morphology, behavior and gene expression, and undergo a number of unique biological processes:




  • Lengthy migration through the developing embryo to the gonadal ridge



  • Erasure of epigenetic information from the previous generation



  • Reactivation of the X chromosome that has been silenced (Barr body) in XX cells.



Migration of Primordial Germ Cells


PGCs proliferate by mitosis, and begin to migrate through the embryonic tissue, completing approximately six mitotic divisions by the time they colonize the future gonad (Figure 3.1).





Figure 3.1 Migration of primordial germ cells in the mouse. PGCs arise at the start of gastrulation, around embryonic Days 7–7.25 (E7–7.5) at the border of the extraembryonic tissue and the epiblast, at the root of the allantois. The PGCs can be identified and distinguished from the surrounding somatic cells by their positive staining for alkaline phosphatase. Expression of the OCT4 transcription factor becomes restricted to PGCs around day E8 and is used as a PGC marker.


Adapted diagram courtesy of J. Huntriss, University of Leeds. Modified from Starz-Gaiano & Lehmann, 2001, with permission from Elsevier Ltd.

Proliferation and migration continue for 3–4 weeks in humans, and during their migration the germ cells and the somatic cells interact together via a number of different types of signals. The PGCs move to the inside of the embryo along with the gut to embed in the connective tissue of the hindgut, migrate through the dorsal mesentery along the hindgut a few days later, and then finally populate the gonadal ridge to form the embryonic gonad. The tissue of the gonadal ridge makes up the somatic (nongamete) part of the gonads. In humans, PGCs can be seen in the region of the developing kidneys (the mesonephros) approximately 4 weeks after fertilization (gestational age 5–6 weeks), and their migration is completed by 6 weeks of gestation. Primordial gonads can be identified on either side of the central dorsal aorta between 37 and 42 days after fertilization (gestational age 8–9 weeks) as a medial thickening of the mesodermal epithelium that lines the coelom (body cavity).




PGCs in the Mouse




  • During their migration in the mouse, the population of PGCs increases from around 100 cells to 25 000 cells by stage 13.5 of embryonic development (E13.5).



  • The genital ridges may secrete a chemotactic substance (probably SDF-1, stromal cell derived factor 1 and its receptor CXCR4) that attracts the PGCs: primordial gonadal tissue grafted into abnormal sites within a mouse embryo attracts germ cells to colonize it.



  • Experiments using gene knockout animal models have identified a number of genes involved in regulating the establishment and migration of PGCs, the signals involved in their movement and their renewal properties (see MacLaren, 2003).



Gonadogenesis: From Primordial Germ Cells to Gametes


The primordial gonadal ridges develop on the posterior wall of the lower thoracic and upper lumbar regions, and in both sexes this undifferentiated mesenchymal tissue forms the basic matrices of the testes and ovaries. At approximately 6 weeks of gestation the developing gonad appears identical in male and female embryos, and remains sexually undifferentiated for a period of 7–10 days. During this period, groups of cells derived from the columnar coelomic (germinal) epithelium surrounding the genital ridge migrate into the undifferentiated tissue as columns to colonize the gonad; these are known as the primitive sex cords. Key morphological changes then start to take place in the gonads, which depend upon whether or not the Sex-determining Region Y (SRY) gene on a Y chromosome is expressed in the cells of the sex cords. These morphological changes result in the formation of:




  1. 1. Genital ducts




    • Wolffian duct = male, precursor of the vas deferens and epididymis



    • Müllerian duct = female, precursor of the uterus, the upper parts of the vagina and the oviducts.




  2. 2. Urogenital sinus.


Knockout mouse technology has identified a number of genes involved in these early stages of gonadogenesis, some of which are outlined in Figure 3.2, and summarized here:




  1. 1. SRY expression is upregulated by only one isoform of Wilms’ tumor gene product WT1(–KTS).



  2. 2. The WT1(–KTS) isoform also upregulates DAX1 expression (which antagonizes development of Sertoli cells). The WT1(–KTS) isoform is therefore considered essential for development of both the male and the female gonad.



  3. 3. The WT1(+KTS) isoform increases the number of SRY transcripts and is required for formation of the male gonad.



  4. 4. WNT4 acts to repress migration of mesonephric endothelial and steroidogenic cells in the XX gonad, preventing the formation of a male-specific coelomic blood vessel and the production of steroids. WNT4 expression is downregulated after sex determination in the XY gonad.



  5. 5. DAX1 may inhibit SRY indirectly by inhibiting expression of male-specific genes that are activated by SF1.



  6. 6. SRY upregulates expression of a related transcription factor, SOX9. SOX9 is required for activation of anti-Müllerian hormone (AMH)/Müllerian inhibitory substance (MIS), which causes regression of the female Müllerian duct.



  7. 7. Dmrt1 is thought to interfere with the action of SOX9. Dmrt1 is a candidate sex-determining gene in birds, carried on their Z chromosome.





Figure 3.2 Genes involved in gonadogenesis; key points are circled, outlining the complex regulatory molecular pathways involved.


Adapted with permission from Clarkson & Harley, 2002.



Sex Determination


A classic experiment by Alfred Jost in the 1940s demonstrated that mammalian embryos castrated prior to differentiation of the testis appear to develop phenotypically as females. This established that the female route of sexual development is the default differentiation pathway, and led the authors to propose the existence of a testis-determining factor (TDF). This has now been established by experiments on early embryos, and by molecular experiments. Genetic studies also suggest that ovarian differentiation and development may be regulated by certain ‘anti-testis’ factors:




  • In XY humans who carry a duplication of part of the small arm of the X chromosome (Xp21) (and in XY mice of certain genetic backgrounds), overexpression of the DAX1 gene causes sex reversal; i.e., these human or mouse individuals develop as females. Therefore, DAX1 can apparently antagonize SRY in a dosage-sensitive manner to cause sex reversal.



  • Wnt4 is also required for female development. Genetic studies in the mouse show that:




    • Wnt4 is initially required for the formation of the Müllerian duct in both sexes.



    • In the developing ovary, Wnt4 suppresses the development of Leydig cells.



    • In Wnt4 mutants, the Müllerian duct is absent, and the Wolffian duct develops further.



Wnt4-mutant females activate testosterone biosynthesis and become masculinized.


Knock-outs of SF1, Lim-9 and Wnt1 genes develop as phenotypic females, in support of Jost’s proposal.



Development of the Testis


After the mesonephros has been populated with primordial germ cells to form the genital ridge, the coelomic epithelium proliferates at a faster rate in male than in female gonads, and the cells penetrate deep into the medullary mesenchyme to form the testis cords. Two different testicular compartments are formed: the testicular cords and the interstitial region. Expression of the SRY gene initiates differentiation of Sertoli cells, and the developing Sertoli cells produce a growth factor, fibroblast growth factor 9 (FGF-9), that is necessary for formation of the testicular cords. At 7–8 weeks of gestation, the testicular cords (precursor of the seminiferous tubules) can be seen in histological sections as protrusions of the cortical epithelium into the medulla; animal experiments indicate that germ cells are not involved in this process.




  • Sertoli cells cluster around the germ cells; peritubular myoid cells surround the clusters and deposit the basal lamina.



  • Sertoli cells secrete AMH/MIS, which suppresses the default pathway that would develop Müllerian ducts as precursors of female sexual anatomy. The Sertoli cells continue to secrete AMH throughout fetal and postnatal life until the time of puberty, when the levels drop sharply.



  • Leydig cells remain in the interstitial region, close to blood and lymphatic systems, and they actively secrete androgens from at least 8–10 weeks of gestation. This capacity to secrete testosterone is essential for continued testicular development and, ultimately, for the establishment of the male phenotype.



  • Testosterone causes growth and differentiation of the Wolffian duct structures (precursor of the male sexual anatomy).



  • Dihydrotestosterone (a metabolite of testosterone) induces virilization of the urogenital sinus and the external genitalia.


The Müllerian duct then regresses, and the Wolffian duct develops further.


By 16–20 weeks of fetal life, Sertoli cells and relatively quiescent prospermatogonial cells lie on a basement membrane within seminiferous cords; these are within a vascularized stroma that also contains condensed Leydig cells, and the entire structure is enclosed within a fibrous capsule, the tunica albuginea. The testes gradually increase in size until the time of puberty, and with the onset of puberty they begin to rapidly enlarge:




  • The solid cords canalize to give rise to tubules, which eventually connect to the rete testis, the vasa efferentia and then the epididymis.



  • Leydig cells significantly increase their endocrine secretion, and intratubular Sertoli cells also increase in size and activity.



  • Prospermatogonial PGCs (gonocytes) in the cords now line the seminiferous tubules as spermatogenic epithelium and begin to divide by mitosis.


After the population has been expanded, the prospermatogonia enter a quiescent non-proliferative phase; they are initially located toward the center of the seminiferous cords, but then migrate to a niche in the periphery where they can begin to function as spermatogenic stem cells (SSC), known as as type A spermatogonia. This SSC population must both self-renew to maintain its numbers and generate progenitor cells that then enter spermatogenesis to produce mature sperm.




Genetic Control




  • SRY, SOX9, WT1, XH2, SF1 and DAX1 are known to be involved in the control of testis determination. Many of these genes have been identified through analysis of cases of sex reversal.



  • The SRY gene is a key switch in male sexual differentiation; it acts only briefly in male embryos to initiate differentiation of the Sertoli cells in the somatic cells of the genital ridge.



  • SRY may function either:




    • As a transcriptional repressor to repress activation of the genes that cause differentiation of the ovary.



    • As a repressor of the factors that repress testis development.



    • Synergistically with SF1 to activate SOX9.




Testicular Descent

The testes develop initially in the upper lumbar region of the embryo and gradually migrate during fetal life through the abdominal cavity and over the pelvic brim. This descent is influenced by hormones secreted by the Leydig cells and involves two ligamentous structures: the suspensory ligament at the superior pole and the gubernaculum at the inferior pole of the testis. As the fetal body grows in size, the suspensory ligament elongates and the gubernaculum does not, so that the position of the testis becomes localized to the pelvis. Between weeks 25 and 28 of pregnancy, the testes migrate over the pubic bone, and reach the scrotum via the inguinal canal by weeks 35–40. As a result of their external position outside the body cavity, the temperature of the testes is approximately 2°C below body temperature, which is optimal for spermatogenesis.


The adult testes contain approximately 200 m of seminiferous tubules, forming the bulk of the volume of the testis. These tubules are the site of spermatogenesis. The round tubules are separated from each other by a small amount of connective tissue that contains, in addition to blood vessels, a few lymphocytes, plasma cells and clumps of interstitial Leydig cells. The tubules are lined by spermatogenic epithelium, which is made up of spermatogonia at different stages of maturation; a cross-section of any normal seminiferous tubule reveals four or five distinct generations of germ cells. The younger generation cells are on the basement membrane, and the more differentiated cells approach the lumen of the tubule. This growth pattern has a wavelike cycle with intermingling of different stages that lie close to each other; any single cross-section of the tubule does not always reveal all generations of spermatogenesis. The tubules rest on a delicate anuclear basement membrane that in turn lies on a connective tissue layer, the tunica propria. The supporting Sertoli cells, which are believed to nourish the germ cells, form a continuous layer connected by tight junctions. These large polymorphous cells have large, pale nuclei and abundant cytoplasm that extends from the periphery of the tubule to the lumen, stretching through the layers of developing germ cells. Mature spermatozoa can be seen attached to and surrounding the Sertoli cells prior to their release (Figure 3.3). A wave of spermatogenesis passes along the tubule, and the process of development from spermatogonium to spermatozoon takes approximately 65 days. A transverse cross-section through the human testis shows tubules containing cells at many different stages in spermatogenesis (in contrast to the rat testis, where every tubule has cells at the same stage). In humans, many seminiferous tubules can be seen that are apparently without spermatocytes and spermatids, a phenomenon that may contribute to the relatively poor efficiency of spermatogenesis.





Figure 3.3 Spermatogenesis in the mammal. Maturation and modeling of the male gamete is regulated by the Sertoli cell.


Modified with permission from Johnson [2018].

Sperm released into the lumen of the seminiferous tubules pass via the rete testis through the ductuli efferentia into the caput epididymis. They traverse the epididymis over a period of 2–14 days, undergoing a number of changes in preparation for fertilization, and are then stored sequentially in the cauda, vas, seminal vesicles and ampullae prior to ejaculation. The seminal vesicles, prostate and urethral glands add glandular secretions to the sperm at the time of ejaculation. Figure 3.4 illustrates the anatomy of the adult mammalian testis.





Figure 3.4 Anatomy of the adult mammalian testis.


Drawing adapted from a number of sources.


Spermatogenesis


In the fetal testis, primordial germ cells differentiate into spermatozoal stem cells, type A (A0 or As) spermatogonia. The process of spermatogenesis is initiated at puberty and continues throughout the reproductive life of the individual. This need for continual production requires a thriving stem cell population, major expansion of progenitor cells, morphological transformation from spermatid to sperm and the acquisition of motility. The entire process is subject to a high level of control and orchestrated organization, with complex interactions between the germ cells, testicular somatic cells, and a number of endocrine and growth factors.


Spermatogenesis can be divided into three well-defined phases, and each phase is associated with a specific type of precursor cell:




  1. 1. Proliferation (mitotic expansion, spermatocytogenesis): spermatogonia



  2. 2. Reduction division (meiotic reduction): spermatocytes



  3. 3. Differentiation (morphological transformation, spermiogenesis): spermatids.


These precursor stem cells line the basement membrane of the seminiferous tubules, have large spherical or oval nuclei, and are connected to each other via intercellular bridges to form a germ cell syncytium. They are in contact with Sertoli cells, which extend from the epithelium into the lumen of the tubules. At puberty, the spermatogonia start to proliferate by mitoses; this is followed by meiosis and a gradual reorganization of cellular components and a loss of cytoplasm.



Spermatocytogenesis



  • At intervals after puberty, stem cells in the germinal epithelium of the seminiferous tubules (type A spermatogonia) replicate their DNA and divide by mitosis.



  • Each mitotic division produces two cells: another type A spermatogonium and a second cell, which enters a pool of undifferentiated spermatogonia – transient amplifying progenitor cells, Aa1, that transition without cell division into type A1 differentiating spermatogonia.



  • These A1 cells undergo five synchronized cell divisions to form A2, A3, A4, Intermediate (In) and B spermatogonia, which then move into the adluminal compartment and start their differentiation by entering into meiosis.



Meiosis

The transition from undifferentiated (A) to A1 spermatogonia represents an irreversible commitment to meiosis, and is controlled by numerous intrinsic factors and by at least one extrinsic factor: retinoic acid (RA), a metabolite of vitamin A (see Griswold, 2016 for review). In the adluminal compartment, the cells undergo two meiotic divisions to form two daughter secondary spermatocytes initially, and eventually four early spermatids. Through a series of different phases, meiosis (reduction division) converts diploid stem cells (spermatogonia) containing 46 chromosomes into haploid gametes, with 23 chromosomes. In the first phase of meiosis, type B spermatogonia (2n:2c) become primary spermatocytes (1n:2c). These cells divide again to become secondary spermatocytes (1n:1c). The cells go through this stage quickly and complete the second meiotic division. After the second meiotic division, the cells are known as spermatids. These cells must now go through a process of maturation (spermiogenesis) in order to finally emerge as mature spermatozoa (1n:1c).



Spermiogenesis

Spermatid differentiation occurs in four stages (Figure 3.5):




  1. 1. Golgi phase



  2. 2. Cap phase



  3. 3. Acrosomal phase



  4. 4. Maturation phase.


Spermatid nuclei now contain haploid sets of chromosomes. Their autosomes continue to direct the synthesis of low levels of rRNA, mRNA and proteins as they enter into their prolonged phase of terminal differentiation, spermiogenesis. During this phase, round spermatid cells are converted into elongated sperm cells with a condensed nucleus and a flagellum. They do not divide again, either by mitosis or meiosis, but must differentiate to acquire functions that will allow them to traverse the female tract and fertilize an oocyte. This differentiation process takes approximately 2 weeks in most species and follows well-defined stages:




  1. 1. Spermatid DNA becomes highly condensed and somatic histones are replaced with protamines.



  2. 2. The acrosome, a sac containing enzymes necessary for oocyte penetration, is constructed from Golgi membranes.



  3. 3. Cytoplasmic reorganization gives rise to the midpiece, which contains mitochondria and associated control mechanisms necessary for motility.



  4. 4. The flagellar apparatus (tail) is formed, which will make the cells motile.



  5. 5. A residual body casts off excess cytoplasm.





Figure 3.5 Developing sperm. A = acrosome; An = annulus; Ax = axoneme; C = centriole; F = flowerlike structures; Fs = flagellar substructures; M = mitochondria; Mp = middle piece; Mt = manchette; Ne = neck; PP = principal piece; R = ring fibers; Sb = spindle-shaped body.


Courtesy of M. Nijs and P. Vanderzwalmen, Belgium.

Sperm modeling is probably regulated by the Sertoli cells, and the cells are moved to the center of the tubular lumen as spermatogenesis proceeds. The rate of progression of cells through spermatogenesis is constant and is not affected by external factors such as hormones. The timing of stored mRNA translation is a major point of control: for example, the protamine1 gene is transcribed in round spermatids, and the resulting mRNA is stored for up to 1 week before it is translated in elongating spermatids. Other mRNAs are stored for only hours or a few days, indicating that there must be a defined temporal program of translational control.




  • Sertoli cells are connected by tight junctions and act as nurse cells during spermatogenesis.



  • Tight junctions restrict the passage of substances from the blood to the lumen of the seminiferous tubules and therefore form a blood–testis barrier. This barrier protects sperm from antibodies circulating in the bloodstream.




Molecular Features of Spermatogenesis




  • The trigger that determines which spermatogonia become committed to meiosis is not known.



  • DNA is transcribed from both diploid (proliferative type A [A0] spermatogonia) and haploid (committed type B [A1] spermatogonia) genomes throughout the process.



  • Only type B spermatogonia undergo differentiation into spermatozoa, and the vast majority of the germ cell cytoplasm is lost during the terminal stages of differentiation, when the spermatids condense into spermatozoa.



  • The reduction process that generates haploid sperm cells takes at least 65 days in humans and involves six successive stages over four consecutive spermatogenic cycles.



  • Note the nomenclature relating to chromosome complement (n) and DNA copy number (c). 1c is the DNA copy number of a haploid gamete, and no gamete is ever tetraploid.




Pathology Affecting Spermatogenesis




  • A number of different pathologies can disrupt the orderly pattern of spermatogenesis, causing immature forms, especially spermatocytes to slough into the tubular lumen. Less frequently, maturation may proceed to the spermatid stage and be arrested there.



  • Any lesion that causes generalized arrest of maturation, or a mixture of maturation arrest and atrophy to a stage preceding spermiogenesis, will result in azoospermia.



  • The tubular epithelium is very sensitive to toxins and to ischemia; damage may result in partial, focal or total obliteration of the spermatogenic epithelium, including the Sertoli cells, while the Leydig cells remain functionally normal.



  • In cases of severe injury, the tubules may be totally destroyed and become hyalinized, or may be replaced by fibrous tissue. Since the whole tubule is destroyed, this disorder is associated with a much reduced testis size, with absence of Sertoli cells resulting in raised serum FSH.



  • Focal lesions can cause oligospermia of varying severity, and patients with focal lesions may have normal levels of FSH in their serum.



Epididymal Maturation

Mammalian spermatozoa leaving the testis are not capable of fertilizing oocytes. They gain this ability while passing down the epididymis, a process known as epididymal maturation. The epididymis is divided into different regions: the caput is the upper third, formed by efferent ductules that are lined by pseudostratified columnar ciliated epithelium (such as is found in nasal and bronchial passages; patients with upper epididymal obstruction often have associated nasal or respiratory problems, as in mucoviscidosis, Young’s syndrome). The vasa efferentia tubules unite to form the single coiled tubule of the corpus, which has flatter, non-ciliated epithelium and microvilli on the luminal surface. It starts to form a muscular wall toward the cauda, where the lumen is wider, and spermatozoa can be stored prior to ejaculation.


During its journey through the different regions of the epididymis, the head of the spermatozoon acquires the ability to interact with the zona pellucida, with an increase in net negative charge. Many antigens with a demonstrable role in oocyte binding and fusion are synthesized in the testis as precursors, and then activated at some point in the epididymis either through direct biochemical modification, through changing their cellular localization or both. Examples of such antigen processing include a membrane-bound hyaluronidase, fertilin, proacrosin, 1,4-galactosyltransferase (GalTase) and putative zona ligands sp56 and p95. The terminal saccharide residues of membrane glycoproteins and lipids also undergo changes in their physical and chemical composition.


Although all the necessary morphological structures for flagellar activity are assembled during spermiogenesis, testicular spermatozoa are essentially immotile, even when washed and placed in a physiological solution. Spermatozoa from the caput epididymis begin to display motility, and by the time they reach the cauda they are capable of full progressive forward motility. Demembranation and exposure to ATP, cAMP and Mg2+ triggers movement, which suggests that the ability to move is probably regulated at the level of the plasma membrane. Transfer of a forward motility protein and carnitine from the epididymal fluid is believed to be important for the development of sperm motility. Since the osmolality and chemical composition of the epididymal fluid vary from one segment to the next, it may be that the sperm plasma membrane is altered stepwise as it progresses down the duct, and motility is controlled by an interplay between cAMP, cytosolic Ca2+ and pH. During maturation, the spermatozoa use up endogenous reserves of metabolic substrates, becoming dependent on exogenous sources such as fructose; at this point they shed their cytoplasmic droplet.



DNA Packaging in Sperm

The amount of DNA in the sperm nucleus (approximately 1 m in length) has to be packaged into a volume that is typically less than 10% of the volume of a somatic cell nucleus; a different mechanism of packaging is required, as illustrated in Figure 3.6.




  • Somatic cell DNA is packaged into nucleosomes by a process of primary compaction that uses histones. A 10-nm fiber is supercoiled into the 30-nm solenoid, and supercoiled again into loops (Figure 3.6A–D. These loops are the major structural form of interphase chromatin.



  • During spermatogenesis, DNA is initially packaged by histones (as in Figure 3.6A–D) but following meiosis, at the secondary spermatocyte stage of spermiogenesis, histones are replaced first by transition proteins and then by protamines (Figure 3.6G). The solenoid structure is replaced by torroids (doughnut shapes), which are in turn supercoiled into torroidal loops. This highly compacted structure shuts down transcription during spermiogenesis. The loop domains shown in Figure 3.6E and J represent the chromatin state in the interphase somatic cell nucleus (Figure 3.6E) and the sperm nucleus (Figure 3.6J). Sperm appear poised for transcription.





Figure 3.6 DNA packaging in somatic cells and spermatozoa.


Image adapted with permission from Ward, 1993.


Gene Expression during Spermatogenesis

Gene expression during spermatogenesis can be subdivided into two distinctive phases:




  1. 1. Prior to meiosis, all stages up to and including the completion of telophase: diploid cells.



  2. 2. During spermiogenesis: late secondary spermatocytes are haploid cells, and the developmental process from this stage onwards is referred to as spermiogenesis.


Genes are expressed from both the diploid and the haploid genomes. DNA transcription is often coordinated with mRNA translation into its protein product (e.g., histones, X-linked lactate dehydrogenase). In spermiogenesis, however, transcription may be shut down well before the protein appears, i.e., the mRNA is translated later (e.g., PGK2, acrosin). As spermatogenesis progresses, the transcripts encoding the same protein differ in size, due to alterations in the length of the mRNA polyA tail. (A similar phenomenon occurs in oogenesis, as described later.)




Control of Gene Expression during Spermatogenesis




  • Between primitive spermatogonial and final mature spermatozoa, cellular chromatin is restructured so that certain genes are repressed, potentiated, or potentiated and transcribed.



  • The gene for phosphoglycerokinase 1 (PGK1), an essential glycolytic enzyme, lies on the X chromosome, and is highly expressed, potentiated and transcribed early in spermatogenesis. As the cells progress into meiosis, the X chromosome becomes progressively inactivated, and during spermiogenesis expression of PGK1 is replaced by expression of an autosomal homologue, PGK2.



  • During spermiogenesis (the haploid stages of spermatogenesis), cellular histones are replaced first by testis-specific histones and then by the transition protein TNP2 and the protamines PRM1 and PRM2. This substitution is a prerequisite for the extremely compact packaging of sperm DNA. These genes are located on chromosome 16.



  • Not all histones are replaced. Human sperm retain approximately 15–20% of their chromatin in a nucleosomal configuration, and we now know that mature sperm cells retain a complex repertoire of mRNAs that may be involved in embryogenesis.


The ultimate aim of the male reproductive system is to parcel the male genetic package, a set of 23 chromosomes, into the head of a single spermatozoon, and deliver this to the female reproductive tract, in the right place at the right time. However, in order to fertilize the oocyte and initiate embryonic development, the spermatozoon must also contribute two epigenetic factors: an oocyte-activating factor and the centrosome or cell division mechanism (see Chapter 4).



Development of the Ovary


The female genital tract develops from the mesonephros; the nephric duct and mesonephric tubules degenerate, and the oviducts and uterus evolve from the paramesonephric duct. This is a transport epithelium, which contains both ciliated and secretory cells: the ampulla develops from a secretory region. In the epiblast of early female embryos, a small number of progenitor cells come under the influence of locally secreted factors that suppress genes responsible for somatic development (e.g., Hox) and permit expression of genes that are specific for germline development, such as Stella and Blimp1 (Saitou et al., 2005). Primordial germ cells containing two X chromosomes are translocated from the genital ridge to the primordial gonad, and these are known as oogonia. The sex cords, instead of penetrating deeply into the genital ridge as in males, condense as small clusters around the PGCs, and these clusters of cells initiate the formation of primordial follicles (Figure 3.7). Cells of the cortical sex cords will form the somatic components of the follicle: granulosa, theca, endothelial cells and supporting connective tissue. Once they reach the gonadal ridge (approximately Days 25–30), the oogonia start to replicate by mitosis for a limited period. Cells from the mesonephros invade to form the ovarian medulla, forcing the germ cells toward the ovarian cortex. Whereas in male embryos spermatogonia do not start to enter meiosis until the onset of puberty, in females the oogonia start to enter into their first meiotic division around the twelfth week of gestation, at the end of the first trimester. In humans, the population of oogonia is estimated to increase from a pool of around 600 000 at 8 weeks to a maximum of 6–8 million at 16–20 weeks. At this stage they become primary oocytes and do not replicate further by mitosis. Oocytes that are not incorporated into follicles degenerate, and thus the number of oocytes is then reduced to around 1–2 million at birth, when the ovary is now populated with its full complement of oocytes.





Figure 3.7 Development of the human ovary from PGC. PGCs travel along the gut (G) mesentery (1) to the gonadal ridge (2), and after proliferation and migration become associated with cortical cords (C, 3). They begin meiosis and become enveloped within follicles (F, 4). Ad = adrenal gland; A = aorta; V = cardinal vein; E = coelomic epithelium; M = mesonephric tubules and duct.


Reproduced with permission from Gosden (1995).

After the oogonia enter meiosis I, they arrest in the diplotene stage of prophase I, after chromatid exchange and crossing-over (diakinesis) have taken place – the last phase of prophase I (see Chapter 1, Figure 1.13). These arrested oocytes are said to be in the dictyate (germinal vesicle) stage. The chromosomes disperse and appear as visible chromosomal threads packaged within a large nucleus, the germinal vesicle (GV). The first meiotic prophase stage can be seen at around 9–10 weeks, and diplotene stage chromosomes are apparent around 16 weeks, during the second trimester of pregnancy. The oocytes remain arrested at this stage until the onset of ovulatory cycles at puberty: subsequent developmental stages that lead to the resumption of meiosis are not completed unless the Graafian (antral) follicle is recruited after puberty. The process of oogenesis, from primordial germ cell to preovulatory oocyte, takes a minimum of 11 years; human oocytes complete meiosis only after fertilization.


The oogonia within the embryonic ovary are initially arranged into clusters called syncytia, which are connected by intercellular bridges. Organelles, mitochondria and other cellular factors are probably exchanged through these connections. These syncytia are programmed to break down on a large scale during fetal life, and this is followed by the formation of primordial follicles. A single layer of pregranulosa cells surrounds a single oogonium; once a complete cell layer has been formed around individual oocytes, the surrounding stromal cells secrete type IV collagen, laminin and fibronectin. These proteins form a thin basement membrane around each cluster of cells, and a discrete population of primordial follicles is formed. Each follicle has an oocyte arrested in prophase I of meiosis, surrounded by a single layer of flattened stromal pregranulosa cells that are linked to the oocyte by gap junctions and other cellular connections. The primordial follicles become localized to the peripheral region of the ovarian cortex and remain there in a quiescent state for many years (Figure 3.8a). In this pool of follicles, each will either undergo a phase of growth and development that lasts approximately 6 months, or will become atretic and die. When they resume their growth after puberty, usually only one oocyte matures and is ovulated per month for the remaining 35 or so years of the reproductive lifespan (Figure 3.8b). Oocytes must complete their growth phase and resume meiosis before they can be fertilized.


Sep 17, 2020 | Posted by in OBSTETRICS | Comments Off on Chapter 3 – Gametes and Gametogenesis

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