Sex determination and germ cells

Two developmental questions concern reproduction directly. How does an embryo set aside the cells that will make eggs and sperm, the germ line that carries genes to the next generation? And how does it decide whether to develop as female or male? The answers vary across animals more than for most developmental processes. Germ cells may be specified by inherited cytoplasm or by signals; sex may be set by chromosomes, by temperature or by social environment. BIOL-201 lesson 7 covered sex chromosomes, sex linkage and the genetics of chromosomal sex determination. This lesson focuses on the developmental mechanisms: how germ cells are specified and migrate, how the gonad chooses between testis and ovary, how that choice is maintained, and how animals without sex chromosomes make the same decision.

The germ line and the soma

In 1892 August Weismann proposed that the germ line, which transmits heredity, is separate from the soma, the body, which dies with the individual. This germ plasm theory explains why changes acquired by the body are not inherited. In most animals the germ line is set aside early, as primordial germ cells (PGCs), which are protected from the differentiation signals around them, migrate to the developing gonads, and only there begin gametogenesis. PGCs share features across species: they are transcriptionally quiet during early stages, they repress somatic gene programs, and they express conserved RNA-binding proteins such as Vasa, Nanos and Piwi.

Two ways to specify germ cells

Germ cells are specified in one of two ways. In preformation, a specialized cytoplasm, the germ plasm, is localized in the egg and inherited by the cells that will become germ cells. Drosophila, C. elegans, Xenopus and zebrafish use germ plasm. In Drosophila, the pole plasm at the posterior of the egg contains Oskar protein, which nucleates the assembly of germ granules containing Vasa, Nanos and other proteins and RNAs. Nuclei that enter the pole plasm bud off as pole cells, the first cells to form in the embryo. Transplanting pole plasm to the anterior of an egg causes nuclei there to form pole cells, which can produce functional gametes if moved to the gonad region. The polar granules of Drosophila and the P granules of C. elegans are examples of biomolecular condensates, membraneless compartments formed by phase separation of proteins and RNAs.

In induction, used by mammals and probably by the common ancestor of animals, germ cells are specified by signals rather than inherited determinants. In the mouse, BMP4 from the extraembryonic ectoderm, together with Wnt3, induces a small group of about 40 epiblast cells, around embryonic day 6.25, to express the transcription factors Blimp1, Prdm14 and Tfap2c. These factors repress the somatic mesodermal program the cells would otherwise follow, reactivate pluripotency genes and begin the epigenetic reprogramming of the germ line. Remarkably, Mitinori Saitou's group showed in 2011 that mouse embryonic stem cells can be converted into PGC-like cells in culture, which form functional sperm and eggs when transplanted, and in 2016 Katsuhiko Hayashi's laboratory completed the entire cycle of mouse oogenesis in a dish. Human PGC-like cells have also been made, though human germ cell specification differs from that of mice, requiring SOX17 rather than Sox2.

Migration and reprogramming of germ cells

PGCs usually form far from the gonads and must migrate there. In mice they arise at the base of the allantois, are carried into the hindgut by the movements of gastrulation, and then migrate actively through the gut mesentery to the genital ridges, guided by the chemokine Cxcl12 (also known as SDF-1) acting through its receptor Cxcr4, and supported by the growth factor Steel (Kit ligand) acting on the Kit receptor. Mice with mutations in Kit or Steel have few germ cells, as well as pigment and blood defects, because the same pathway supports melanocyte and blood stem cells. PGCs that wander off course normally die, but rare survivors can form germ cell tumors such as teratomas, which contain mixtures of differentiated tissues.

During and after migration, mammalian PGCs undergo dramatic epigenetic reprogramming. Genome-wide DNA methylation falls to very low levels, and the parental imprints described in lesson 2 are erased. New imprints are then established according to the sex of the embryo: paternal imprints in the developing male germ line and maternal imprints in the female germ line. This resetting ensures that each generation transmits imprints appropriate to the parent of origin. Errors in germ line reprogramming contribute to imprinting disorders and may allow some epigenetic marks to escape erasure, a topic of current research on transgenerational inheritance.

The bipotential gonad

In mammals, the gonad begins as a bipotential structure, the genital ridge, formed from intermediate mesoderm and coelomic epithelium. Until about the sixth week of human development, the gonads of XX and XY embryos look identical, and both sexes have two pairs of ducts: the Wolffian ducts, which can form male reproductive tracts, and the Müllerian ducts, which can form oviducts, uterus and upper vagina. Supporting cells in the gonad, the precursors of Sertoli cells in testes or granulosa cells in ovaries, make the decision. In 1947 Alfred Jost removed the gonads from rabbit embryos and found that all developed female reproductive tracts regardless of their chromosomes, showing that the testis directs male development and the female pathway was long thought of as a default. Modern work shows that ovary development is also an active genetic program.

SRY and the testis pathway

In 1990 Peter Goodfellow, Robin Lovell-Badge and colleagues identified SRY (sex-determining region of the Y) as the testis-determining gene in humans and mice. XY people with mutations in SRY develop as females (Swyer syndrome), and XX people carrying a translocated piece of Y containing SRY develop as males. The definitive experiment came in 1991, when Koopman and colleagues introduced a 14-kilobase fragment containing mouse Sry into XX mouse embryos and some developed as males, although they were sterile because they lacked other Y-chromosome genes needed for sperm production.

SRY acts briefly, in mice for only about two days around embryonic day 11.5, and its main target is SOX9, a related transcription factor. SRY, with the steroidogenic factor SF1, binds an enhancer of Sox9 called TESCO and, in humans, a far upstream enhancer region. Once activated, SOX9 maintains its own expression through positive feedback loops involving FGF9 and prostaglandin D2, and it drives supporting cells to become Sertoli cells. Sertoli cells organize the testis cords, recruit cells to form Leydig cells, which make testosterone, and secrete anti-Müllerian hormone, which causes the Müllerian ducts to regress. Duplications of SOX9 can cause testis development in XX people even without SRY, and loss of SOX9 causes campomelic dysplasia, a skeletal disorder often accompanied by XY sex reversal.

The ovary pathway and mutual antagonism

In XX gonads without SRY, supporting cells activate a different program. The secreted protein R-spondin 1 (RSPO1) and Wnt4 activate β-catenin signaling, and the transcription factor FOXL2 promotes granulosa cell identity. The testis and ovary pathways actively antagonize each other: SOX9 and FGF9 repress Wnt4 and β-catenin, while β-catenin and FOXL2 repress Sox9. The outcome is a bistable switch that settles firmly into one state. Strikingly, the decision must be maintained throughout life. When Foxl2 was deleted in adult female mice in 2009, granulosa cells transdifferentiated into Sertoli-like cells and began making testosterone; conversely, deleting the transcription factor Dmrt1 in adult male mice caused Sertoli cells to become granulosa-like. Sex determination in mammals is therefore not a single event but an ongoing balance.

Other chromosomal systems

Birds use a ZZ/ZW system, in which females are the heterogametic sex. The Z chromosome carries DMRT1, and two copies in ZZ embryos promote testis development, while the single copy in ZW embryos is insufficient; knocking out one copy of DMRT1 in ZZ chickens produces ovaries. Bird cells also show cell-autonomous sex identity: chickens that are half ZZ and half ZW, gynandromorphs, are visibly male on one side and female on the other, which suggests that each cell's chromosomes influence its sexual characteristics directly, not only through hormones.

In Drosophila, sex depends on the number of X chromosomes. Embryos with two X chromosomes activate the gene Sex-lethal (Sxl) early in development, because X-linked signal proteins reach a threshold; once activated, Sxl protein maintains its own production by controlling the splicing of its own pre-mRNA. Sxl then controls a cascade of alternative splicing, through transformer to doublesex, producing female-specific Doublesex protein in XX flies and male-specific Doublesex in XY flies. Sxl also prevents the male-specific dosage compensation complex from forming in females; in males this complex doubles transcription from the single X. Doublesex and mammalian DMRT1 share a DNA-binding DM domain, a sign that some components of sex determination are ancient even though the upstream triggers differ.

Temperature-dependent sex determination

In many reptiles there are no sex chromosomes, and sex is set by the temperature at which the egg incubates during a sensitive window in the middle third of development. In most turtles, warm temperatures produce females and cool temperatures produce males (pattern Ia); in crocodilians and some turtles and lizards, females develop at both high and low extremes, males at intermediate temperatures (pattern II). The pivotal temperature, at which a 1:1 sex ratio is produced, is often around 29 °C in sea turtles. In 2018, a study of the red-eared slider turtle showed that the chromatin-modifying enzyme Kdm6b, expressed more at cool temperatures, removes repressive marks from the Dmrt1 gene, promoting testis development. Upstream, temperature appears to act through calcium influx and the transcription factor STAT3 in the gonad.

Temperature-dependent sex determination makes some species vulnerable to climate change. A 2018 study of green sea turtles nesting on the northern Great Barrier Reef found that more than 99 percent of recently hatched turtles were female, because sand temperatures have risen. Conservation programs shade nests or move eggs to cooler hatcheries. The loggerhead and leatherback sea turtles that nest along the beaches of the southeastern United States and that forage in Canadian Atlantic waters face the same pressure. Some reptiles show more complex interactions: the Australian central bearded dragon has ZZ/ZW chromosomes, but at high temperatures ZZ embryos develop as females, overriding the chromosomes.

import math
# Hatchling sex ratio under temperature-dependent sex determination (pattern Ia).
# p_female(T) = 1 / (1 + exp(-(T - Tpiv) / s)); nest temperatures vary around a mean.
Tpiv, s = 29.2, 0.6           # pivotal temperature and transition width, degC (illustrative, sea-turtle-like)
def p_female(T):
    return 1 / (1 + math.exp(-(T - Tpiv) / s))
def nest_ratio(mean_T, sd_T=1.0, n=401):
    # average over a normal distribution of nest temperatures (simple numerical integration)
    tot = w_sum = 0.0
    for i in range(n):
        T = mean_T - 4 * sd_T + 8 * sd_T * i / (n - 1)
        w = math.exp(-0.5 * ((T - mean_T) / sd_T) ** 2)
        tot += w * p_female(T); w_sum += w
    return tot / w_sum
print("mean nest temp (degC)   share of hatchlings female")
for mean_T in (27.5, 28.5, 29.2, 30.0, 31.0, 32.0):
    print(f"        {mean_T:4.1f}                 {100 * nest_ratio(mean_T):5.1f}%")
# temperature needed to bring a 90%-female beach back to 50% by shading
target = next(t / 100 for t in range(3200, 2700, -1) if nest_ratio(t / 100) <= 0.5)
print(f"cooling nests to a mean of about {target:.1f} degC restores a 1:1 ratio")

mean nest temp (degC)   share of hatchlings female
        27.5                  12.0%
        28.5                  31.2%
        29.2                  50.0%
        30.0                  71.2%
        31.0                  89.3%
        32.0                  97.0%
cooling nests to a mean of about 29.2 degC restores a 1:1 ratio

The model shows how steep the response can be: because the transition from all-male to all-female spans only a few degrees, a warming of 1 to 2 °C in mean nest temperature can shift a beach from balanced to strongly female. The real situation is more complicated, because the sensitive period is only part of incubation, metabolic heating raises temperatures in the center of large clutches, and temperature affects hatching success and the fitness of hatchlings as well as their sex.

Sex change in adults

Some fish change sex as adults in response to social cues. Clownfish live in groups dominated by a breeding pair, with the female the largest; if she dies, the male becomes female and the largest non-breeder becomes the new male. The bluehead wrasse shows the reverse: females can become males within days when the dominant male is removed, beginning with behavioral changes within hours, followed by changes in gonads. Such sequential hermaphroditism shows that the gonad's identity can be plastic in some vertebrates throughout life, an exaggerated version of the maintenance seen in mice lacking Foxl2 or Dmrt1.

Differences of sex development in people

In humans, variations in the genes and hormones of sex development cause differences of sex development (DSD), a term adopted in a 2006 consensus statement to replace older terminology. They include variations in sex chromosome number, such as Turner syndrome (45,X) and Klinefelter syndrome (47,XXY); variations in gonad development, such as Swyer syndrome in XY people with SRY mutations; and variations in hormone production or response, such as complete androgen insensitivity syndrome, in which XY people with mutations in the androgen receptor develop female external anatomy with internal testes, and congenital adrenal hyperplasia, in which XX people exposed to excess adrenal androgens may have masculinized external genitalia. Clinical care has shifted toward multidisciplinary teams, delaying irreversible surgery where possible and involving patients in decisions. These cases show that biological sex involves several developmental layers, chromosomes, gonads, hormones and the responses of tissues to them, which usually align but do not always.