Lecture 10: Sex Determination and Gametogenesis PDF

Lecture 10: Sex determination and gametogenesis Sex determination in Drosophila Like mammals, Drosophila use an XY sex-determination system. Unlike mammals, it is not the presence of a Y chromosome that specifies the sex, but the number of X chromosomes (dosage). One X in a diploid cell =...

Lecture 10: Sex determination and gametogenesis Sex determination in Drosophila Like mammals, Drosophila use an XY sex-determination system. Unlike mammals, it is not the presence of a Y chromosome that specifies the sex, but the number of X chromosomes (dosage). One X in a diploid cell = male Two Xs in a diploid cell = female XO mammals = sterile females (no SRY gene and no Y chromosome); XO flies = sterile males (only one X chromosome) Sex-lethal gene (Sxl) The X chromosome encodes four transcription factors (SisA, Scute, Runt, and Unpaired) that activate the X-linked gene Sex-lethal (Sxl). Sxl is an RNA splicing factor that initiates a cascade of RNA processing events that eventually lead to the expression of a female sexual phenotype.  Functional Sxl is made early in development in XX females Transcription from the early promoter excludes exon 3. The X chromosome encodes four transcription factors (SisA, Scute, Runt, and Unpaired) that activate the early promoter only if they accumulate in high enough concentrations. They only reach this threshold in XX embryos.  Functional Sxl is made early in development in XX females Sxl that is made from the early promoter is spliced in a manner such that exon 3 is absent. Exon 3 contains a stop codon. This means early Sxl is complete and fully functional in early XX embryos.  Early Sxl ensures the formation of more functional Sxl in females Later in development, the late promoter becomes active. By default, transcription from the late promoter will include exon 3. In XX embryos, the Sxl protein that was already made regulates its own splicing to produce the “female functional Sxl”. Early Sxl ensures the formation of more functional Sxl in females Early development Later development Targets of Sxl Three major targets: Sxl itself Msl2 – controls X chromosome dosage compensation Sxl inhibits the translation of Msl2 in females. Msl2 is translated in males (XY) to hyperactivate the single X chromosome to equalize its output to that of two female X chromosomes. If Sxl is nonfunctional/mutated in a cell with two X chromosomes, the result will be cell death because Msl2 will be translated à hyperactivation of both X chromosomes This is where the name Sex Lethal comes from Transformer (tra) – the next gene in the sex determination cascade Transformer (Tra) Loss of function mutations in this gene cause female-to-male transformations (hence the name). tra pre-mRNA is transcribed in both males and females. Females: Sxl splices a female-specific isoform (197 amino acids). Males: No Sxl. A truncated version of the Tra protein is made because there is a stop codon in exon 2. Transformer protein functions with a co-factor (Tra2) to create a splicing enhancer complex that regulates splicing of a gene called Doublesex (dsx). Transformer (Tra) Doublesex (Dsx) The Dsx gene is expressed in both male and female Drosophila, but it is spliced differently depending on the presence or absence of Tra/Tra2. Dsx is a transcription factor. Females: Tra/Tra2 splice the dsx pre-mRNA to create a female-specific Dsx protein with female-specific domains (DsxF). Female-specific Dsx interacts with other proteins to activate female-specific genes. Males: No Tra/Tra2. dsx is spliced to create a male-specific Dsx protein with male-specific domains (DsxM). Male-specific Dsx interacts with other proteins to activate male-specific genes. Doublesex (Dsx) Practice problem Without looking at the chromosomes, describe how you could determine if a Drosophila embryo was male or female (if there were no outward differences in appearance between them). Environmental sex determination In certain crustaceans, reptiles, and annelids, the sex is red-eared slider turtle determined by environmental factors (not sex chromosomes) – e.g. temperature, daylight, location, presence of other members of the species, water availability Trachemys scripta (red-eared slider turtle): below 28℃ - male, above 31 ℃ - female Environmental sex determination What is the mechanism of ESD? Remember: male birds, fish, and frogs do not have a Sry gene. Sox9 is activated by Drmt1 in males. drmt1 expression in T. scripta is increased at male-determining temperatures (25- 28℃) and is decreased in female- determining temperatures (30-33℃). What regulates dmrt1 expression? Ge et al. 2017. Dmrt1 induces the male pathway in a turtle species with temperature- dependent sex determination. Development. Environmental sex determination Kdm6b mRNA Drmt1 mRNA expression is inhibited by the stained green presence of H3K27me3. H3K27me2 is removed by an enzyme called KDM6B. Expression of KDM6B increases in low temperatures. If KDM6B is present à H3K27me2 surrounding the Drmt1 gene is removed à Drmt1 expressed à Sox9 activated à males are formed Environmental sex determination High (female-promoting) temperatures cause the activation of a calcium ion transporter in gonadal precursor cells. Calcium ion influx leads to the phosphorylation of the STAT3 transcription factor. Phosphorylated STAT3 inhibits KDM6B.  Red = northern Study of C. mydas (green sea Great Barrier Reef turtle). (warmer sand) Blue = southern Turtles originating from the Great Barrier Reef northern Great Barrier Reef (cooler sand) showed extremely female- biased sex ratios (99.1% of. juveniles, 99.8% of subadults, and 86.8% of adults being female). The proportion of females has increased in recent decades. Turtles originating from the southern Great Barrier Reef showed moderately skewed sex ratios (67.8% of juveniles, 64.5% of subadults, and 69.2% of adults being female). Jensen et al. 2018. Environmental Warming and Feminization of One of the Largest Sea Turtle Populations in the World. Current Biology. Gametogenesis in mammals Gametogenesis: differentiation of germ cells into gametes Primordial germ cells (PGCs): bipotential precursors of oocytes and sperm; if they reside in ovaries they become oocytes, if they reside inside testes, they become sperm. Gametogenesis in mammals PGCs do not form inside the gonads. In Drosophila and mammals, PGCs form in the posterior part of the embryo and migrate into the gonads. Transcription and translation are shut down in the PGCs while they migrate to the gonads. PGC migration Quality control mechanism. PGCs that are unable to respond to migration cues will die by apoptosis. PGCs enter the hindgut and travel to the genital ridges (bipotential gonad precursor), dividing by mitosis as they migrate. PGCs are surrounded by a traveling niche of cells that secrete stem cell factor (SCF). This niche promotes the persistence, division, and movement of the PGCs to the genital ridge. PGC differentiation in the gonads Once in the gonad, PGCs are directed to either undergo oogenesis or spermatogenesis by the gonad. The timing of meiosis is a fundamental difference between oocytes and sperm in mammals. Females: meiosis begins in the embryonic gonads Males: meiosis begins at puberty The transcription factor Stra8 is a “gatekeeper” for meiosis. Stra8 promotes DNA replication and meiosis. Stra8 and meiosis Female germ cells within the ovaries: Stra8 is upregulated in female germ cells by Wnt4 and retinoic acid (RA) which is secreted by the adjacent kidney. Stra8 induces meiosis. Stra8 and meiosis Male germ cells within the testes: RA produced by the kidney is degraded by the enzyme Cyp26b1 (one of the genes upregulated by Sox9). No meiosis during embryogenesis. Puberty: RA is synthesized by the Sertoli cells and Stra8 is expressed in sperm stem cells à meiosis. Meiosis Meiosis serves two purposes: Halve the number of chromosomes in a cell to allow reproduction. Generate new combinations of alleles through recombination between paternal and maternal chromosomes. Meiosis I Sister chromatids are attached by a Meiosis I segregates common kinetochore. homologous chromosome pairs and creates two haploid daughter cells containing chromatid pairs. Homologous recombination occurs between prophase I and metaphase I, when the cell induces double-strand breaks in the DNA after the paternal and maternal chromosomes line up with each other. Meiosis II Meiosis II segregates the two sister chromatids. The end result of meiosis II is four haploid daughter cells with unreplicated chromosomes. Spermatogenesis in mammals Spermatogenesis begins at puberty and occurs between Sertoli cells inside the seminiferous tubules. Spermatogenesis in mammals Three major phases: 1. Proliferative phase – sperm stem cells (spermatogonia) multiply by mitosis – type A spermatogonium 2. Meiosis 3. Spermiogenesis – postmeiotic shaping where round spermatids eject most of their cytoplasm and become mature sperm Proliferative (mitotic) phase When mammalian PGCs arrive at the genital ridge and become incorporated into the sex chords that will become the seminiferous tubules, they are called gonocytes. Mitotic proliferation of the gonocytes after birth produces type A spermatogonia. Two types of type A spermatogonia: single and paired. A population of single Type A spermatogonia will maintain sperm production throughout life. Single spermatogonia can divide to self-renew or being the process of sperm differentiation by dividing into paired cells, which are linked by cytoplasmic bridges. Proliferative (mitotic) phase Type A spermatogonia differentiate into type B spermatogonia through a series of 5 mitotic divisions. Type B spermatogonia are linked by cytoplasmic bridges. The meiotic phase Type B spermatogonia contain high levels of Stra8. Type B spermatogonia divide once more (mitosis) to generate primary spermatocytes. mitosis Each primary spermatocyte undergoes the first meiosis I meiotic division to yield a pair of haploid meiosis II secondary spermatocytes. They complete the second division of meiosis to generate spermatids. spermatids Spermatids are still connected to each other via their cytoplasmic bridges. The meiotic phase Which cells in the figure are diploid? mitosis meiosis I meiosis II Which cells in the figure are haploid? spermatids Oogenesis in mammals Mammalian oogenesis needs to: Produce a haploid gamete Prepare a gamete that can support a zygote as it develops into an embryo Ovulated human eggs are the largest cell in the human body. Oogenesis in mammals – four stages 1. Proliferation: ~1000 PGCs reach the genital ridge. Within the developing ovary, PGCs divide rapidly from the 2nd à 7th month of gestation to generate ~7 million oogonia. Most oogonia die after this stage. Oogenesis in mammals – four stages 2. Meiosis I (~5th month of gestation) + dictyate resting stage: the surviving oogonia begin the 1st meiotic division under the influence of retinoic acid (RA) and become primary oocytes. Primary oocytes do not complete meiosis I. They are paused in prophase I. This resting phase (dictyate resting stage) lasts from 12-40 years. At birth, females have ~2 million primary oocytes paused at prophase I within the ovaries. Oogenesis in mammals – four stages 3. Ovulation (begins at puberty): primary oocytes resume meiosis I to become secondary oocytes and they begin meiosis II. Once a month there is a surge of luteinizing hormone (LH) released by the pituitary gland which permits meiosis I to be completed. A few primary oocytes will begin the maturation journey but only one will mature into a secondary oocyte. Secondary oocytes begin meiosis II and pause at metaphase II. This mature secondary oocyte is ovulated and waits for fertilization. Oogenesis in mammals – four stages 4. Fertilization: when the sperm meets the egg in the oviduct, enzymes from the sperm cause the release calcium ions in the egg that activate proteins required for the oocyte to complete meiosis II. The secondary oocyte becomes an ovum. Oogenic meiosis Goal of oogenic meiosis: minimize loss of cytoplasm to allow the oocyte to meet the demands of a growing zygote To minimize cytoplasm Polar body loss, the meiotic spindle doesn’t localize to the center of the dividing cell. The smaller cell becomes a polar body which is eventually degraded. Oogenesis First polar body Second polar body Unlike spermatogenesis in which 1 diploid primary spermatocyte gives rise to 4 haploid sperm, in oogenesis 1 diploid primary oocyte gives rise to 1 mature ovum/oocyte.