Molecular & Developmental Biology (BIOL3530)

With Dr. Brian E. Staveley
Department of Biology
Memorial University of Newfoundland

Germ Cells, Fertilization and Sex

Development of germ cells
Germ fate is specified by germ plasm.
In Drosophila, the egg contains large polar granules contain RNA's & proteins.
Exposure to UV will destroy the pole plasm and germ cells will not develop.
If pole plasm is transplanted to the anterior of another embryo, pole cells will develop there & will develop normally when transplanted to the posterior of a developing embryo.
Pole plasm is localized at the posterior end of the Drosophila egg through the action of maternal genes (such as oskar).
In Xenopus, distinct yolk-free patches aggregate in the vegetal pole and are distributed into the pre-germ cells.
In C. elegans, P granules and the PIE-1 protein are sequestered to the pre-germline.

Germ cell sex determination may depend on cell signals and genetic constitution.
In the mouse, the precursors of the germ cells (PGCs) are induced in the proximal epiblast by signals from the extra-embryonic ectoderm.
During gastrulation, these move to the posterior, above the primitive streak to form a cluster in which the centrally located cells become specified.
Once specificed, the primordial germ cells migrate to the gonads,
In the mouse embryo,  pre-germ cells enter the genital ridge (where the gonads form) and continue dividing.
In the female, cells enter meiotic prophase and arrest until the mouse matures.
In the male, the cells continue to divide but eventually arrest in the G1 phase.
After the mice are born, the cells start dividing again and enter meiosis with maturity.
Mouse germ cells that enter meiosis before birth become eggs and those that enter later become sperm.
Mouse germ cells that fail to enter the genital ridge, start to develop as oocytes (in both males and females) but later development is abnormal.
Differentiation of the germ cells depends upon a reduction in the number of chromosomes (meiosis) but oogenesis and spermatogenesis have different approaches.
Note: In the formation of oocytes, the timing of meiosis (and the formation of polar bodies) varies in different organisms, and is completed in some organisms only after fertilization.
In humans, the number of oocytes decline with age via apoptosis (6-7 million in the early fetus, 400,000 oocytes at puberty): 400 to 500 are released throughout a lifetime.
Both maternal and paternal genomes required for normal mouse development.
Imprinting of genes, such as IGF-2 in mouse, are at least partially responsible for this requirement.

Fertilization involves cell-surface interactions between egg and sperm.
The sperm has to pass several barriers to enter the egg.
For fertilization of mammalian eggs, the sperm first passes through a layer of cumulus cell embedded in hyaluronic acid aided by the hyaluronidase actvity on its surface.
The 2nd layer is the zona pellucida, a layer of glycoproteins.
The acrosomal reaction (release of enzymes in the sperm head) is mediated by interaction of the ZP3 species-specific receptor and adhesion molecules in the sperm head.
The acrosome releases acrosin (a protease) and an acetylglucosaminidase (which degrades glycoprotein side-chains).
The sperm surface which contains proteins (i.e. fertilin) that can bind the egg's surface are exposed during the acrosomal reaction.
Fertilin binds an intergrin-like receptor of the egg plama membrane to initiate sperm-egg fusion.
In some invertebrates (i.e. sea urchins), an actin filament-driven acrosomal projection allows the sperm and egg to meet through a coat of jelly.
Changes in the egg membrane at fertilization to block polyspermy include, in the sea urchin, depolarization and the release of cortical granules.
Only one sperm may enter an egg.
To prevent polyspermy, enzymes that prevent other sperm from binding to the zona pellucida are released.

At fertilization a number of events occur to activate development (such as increase in protein synthesis, structural changes [cortical rotation]).
Main event is the completion of meiosis, fusion of the nuclei to form a diploid zygotic genome and entry into mitosis.
A calcium wave initiated at fertilization results in egg activation.
The sharp increase in calcium initiates the cell cycle by acting upon proteins that control the cell cycle.
The Xenopus egg is kept in metaphase II by maturation-promoting factor (MPF) a complex including cyclin.
The calcium wave activates a kinase which results in the degradation of cyclin which allows the meiosis to finish & the nuclei to fuse.

Determination of Sexual Phenotype
The early male and female embryos are very similar and differentially develop in latter stages.
Even in some vertebrates, gender is not always chromosome dependent (i.e. temperature at which the alligator embryo develops determines gender and some fish can change sex depending on environment).
In mammals, sex-determining region on the Y chromosome (Sry), once known as the testes-determining factor encodes a transcription factor that specifies malesness.
Translocation of the Sry region to the X results in XX males and Sry alone injected into XX mouse eggs produce males.

Mammalian sexual phenotype is regulated by gonadal hormones
All mammals begin development as gender neutral, the presence of the Y chromosome induces testis development that produce hormones that switch the development of somatic tissues into the male pathway.
This means that the sex of only the gonads is genetically determined but the rest of the cells are neutral (whatever their chromosome complement is).
Their fate depends upon hormones.
The mesonephros (embryonic kidney) contribute to both male and female reproductive organs.

Wolffian & Mullerian ducts
On the sides of the mesonephros are the Wolffian ducts and Mullerian ducts that open into the cloaca.
In females (in the absence of the testes), the Mullerian ducts develop into the oviducts (Fallopian tubes) and the Wolffian ducts degenerate.
In males, the Wolffian duct becomes the vas deferens.
The genital region differentiates after gonad development with the action of the gonadal hormones.

In mammals, signals from the gonad control germ cell identity as either egg or sperm.

Drosophila sex determination

In Drosophila,
1)  XY germ cells transplanted to a female enter the embryo and
2)  XX germ cells transplanted into a testis both develop as non-functional sperm to demonstrate both cell autonomy and environmental signals.

Gynandromorphs are genetic mosaics in which one X is lost in half of the organism(ie. left XX is female and right XO is male).

The primary sex-determining signal is the number of X chromosomes.
The Sex-lethal (Sxl) protein acts as a stable binary genetic switch.
The presence of two X chromosomes results in the production of Sxl .
The presence of Sxl results in the proper splicing of the tra mRNA production of the transformer protein.
Downstream, male and female versions of the double sex gene product are made by sex-specific splicing of  dsx mRNA.
Tra protein (plus Tra2) leads to the splicing of the female dsx mRNA.
The male dsx mRNA is the default product and is made in the absence of female signaling.

In C. elegans, the differences between the male and the hermaphrodite somatic sex determination are controlled by another binary switch mechanism.
The germ cell identity of C. elegans is passed upon timing in the hermaphrodite, as sperm is made initially (and stored) and eggs are made later.

Various strategies are used for dosage compensation of X-linked genes.
The imbalance of X linked genes between males and females must be corrected (dosage compensation).
Mammals achieve this by inactivating one of the two female X chromosomes after the blastocyst has been implanted in the uterine wall.
The inactive X can be seen in the nucleus as a Barr body.
Xist, is a genetic switch, which produces an RNA that interacts with the "X  inactivation region" of the X chromosome.
In Drosophila, the opposite approach is used when Sxl is off, transcription from the single X chromosome is doubled (translation increases as well).

As a gene controlling coat colour in cats resides upon the X-chromosome, X-inactivation leads to very obvious mosaicism in heterozygous females.
Other mammalian females, including humans, are less obvious in their mosaic patterning due to X-inactivation.

email me at bestave@mun.ca