Developmental Biology BY1101 P. Murphy Lecture 3 The first steps to forming a new organism Descriptive embryology I

• Germ cell formation / gametogenesis And • Fertilisation

Why bother with sex? In terms of the efficiency of producing new individuals it is very wasteful- the need for two types of individual, the elaborate processes to produce germ cells and fuse them. So why did it evolve? What are the advantages of sex? It allows genetic variation between individuals → genetic variation in the population. In asexual reproduction the offspring are genetically identical to the parent.

o Variation allows the population to adapt to changing conditions. o Variation is the driving force for evolution o And necessary for a healthy adaptive population

How widespread is sex? Meiosis and sexual reproduction only occur in a small number of lineages on the tree of life. e.g.

Bacteria- only asexual reproduction Most algae, fungi and some land plants - asexual and sexual

reproduction. Yeast- mostly by binary fission- but can undergo sexual reproduction- especially if under stress.

Animals- mostly sexual reproduction but some exceptions- hydra can reproduce by budding

Even an exception among vertebrates- the guppy (fish). But sex is predominant among multicellular organisms and very successful - e.g. insects with millions of species.

Campbell and Reece Fig. 13.5 Fig 13.5 shows an overview of what happens to the genetic compliment during sexual reproduction. It shows the example of the human but the principle is the same for all organisms that reproduce sexually. Basically the genetic compliment of the adult organism is in this case 2n - where each individual has two copies of each chromosome and therefore two copies of the chromosome set and two copies of most genes. This is said to be a diploid compliment or 2n- Note in the figure how the n values change. In the production of germ cells- eggs produced in the ovaries in the case of the female and sperm produced in the testes in the case of the male, a process of cell division (meiosis) that halves the genetic compliment takes place. The germ cells - ovum or egg and sperm- therefore have a genetic compliment of one half of the adult or n- called haploid. So the female parent gives half their genes- one copy of each- and the male parent gives half their genes - one copy of each, to each of the gametes or germ cells. Each of the ova produced by an individual female parent will have a different compliment of genes because the partitioning of the chromosomes to the ova is random. The same goes for the male parent. So when fertilisation happens and a single sperm fuses with a single ovum- the diploid genetic compliment is restored and a new individual is set to emerge. Another fertilisation event, even with the same parents, will involve a different sperm and different ovum and a different unique compliment of genetic

material. Both zygotes will contain 50% of the genes of the female parent and 50% of the genes of the male parent- but a different 50% in each case- so a whole new genetic make up. The only exception is the rare event of monozygotic (identical) twins where the zygote splits into two after a single fertilisation. In non-identical twins -dizygotic - where two separate ova are fertilised by two separate sperm at the same time- the twins are as related as any other pair of sibs and share 50% of their genes on average. From the point of view of this module what is important is that the new individual is set to emerge through the processes of development. A summary of the genetics involved Germ cells contain a random distribution of half the chromosomes of the parent (n) Fusion of germ cells restores the full compliment of genes (2n). ⇒ offspring share 50% of their genes with each of their parents- but which 50% is randomly determined - so siblings will each have 50% of the genes of each parent- but a different 50%. ⇒ siblings share on average 50% of their genes. But the shared genes between siblings could be as low as 0% or as high as 100%- 50% on average The exception is monozygotic twins who share 100% of genes because a single zygote splits in two after fertilisation. Dizygotic twins result from two ova being fertilised separately (multiple ovulation) and share the same as other sibs- average 50% This is what happens usually in organisms with multiple births at the same time- this arises because of multiple ovulation. A variety of Sexual life cycles- in the different life cycles below, different proportions of the cycle are in the haploid (n) and diploid (2n) state. In animals (a below) most time is spent in the diploid state- only sperm and egg are haploid. From fig 13.6 A. Diploid dominant- found in animals B. Alternations of generations Plants and some algae

C. Haploid dominant Most fungi and some protists Whatever the life cycle, genetic variation is the common outcome of sexual reproduction. A general overview of meiosis is given in Campbell and Reece Fig. 13.7 Meiosis I separates the homologous chromosomes and meiosis II separates the sister chromatids Gametogenesis in both sexes is based on meiosis but the details differ in the male and female animal. Here as an example we look at gametogenesis in humans. Spermatogenesis- the production of mature sperm is a continuous process in the adult male. It ccurs in the seminiforous tubule of the testes. In the embryonic testes the primordial germ cells- or the stem cells that will give rise to all sperm, differentiate – these are called the spermatogonia. Mitotic divisions that make more of these stem cells continue throughout the life of the male. To form sperm the spermatogonia undergo two meiotic cell divisions –forming primary and secondary spermatocytes respectively. Both are produced in the seminiferous tubule with the more mature cells located closer to the lumen. Following meiosis the haploid spermatids then differentiate into mature, motile sperm released into the lumen. This last step is not a division but a differentiation- called spermiogenesis- distinct from spermatogenesis which is the whole process. Spermatogenesis is shown in fig 46.12 (Campbell and Reece) Sperm structure From Campbell and Reece Fig 46.12 Sperm structure is very consistent throughout the animal kingdom. The structure is very important for function- as you will see in the process of fertilisation. Oogenesis is shown in Campbell and Reece Fig 46.12 Oogenesis begins already in the developing ovary of the female embryo, with the production of primordial germ cells or oogonia- these are produced by mitosis and are the stem cells of the future eggs. The oogonia give rise to the primary oocytes by initiating the first meiotic division but they arrest in prophase. These primary oocytes are present in

the ovary of the female at birth, each contained within a protective follicle- they remain quiescent like this until puberty when hormonal release causes their maturation. In humans generally only one ovum continues to mature in each cycle- so normally only one offspring born- dizygotic twins would result from a double ovulation- animals with multiple births would of course mature and release multiple eggs simultaneously. At puberty then Follicle stimulating hormone periodically stimulates follicles to grow and induces the primary oocyte to complete meiosis I- this is an unequal division- producing a single secondary oocyte and extruding the extra genetic material in a polar body. the secondary oocyte arrests in metaphase of meiosis II and is released as an ovum during ovulation in this arrested state. In humans, meiosis II is not completed until a sperm fertilises the ovum at which point a second polar body containing the excess genetic material is extruded. In other animals the sperm may enter the ovum earlier, or later or at a similar stage to humans. The ruptured follicle is left behind after ovulation and degenerates. The belief for many years has been that humans, like all female mammals are born with their full compliment of primary oocytes- that no new primary oocytes are generated following birth. This view has recently been challenged by the finding of multiplying oogonia in the adult female mouse- so this may not be fully true. Oogenesis differs from spermatogenesis in 3 major ways: 1. Cytokenesis is unequal in meiosis- most cytoplasm going to a single daughter cell (oocyte)- goes on to form the ovum. The polar bodies degenerate.

Why? Cytoplasm is needed to support the future zygote. Eggs- largest cells in animal kingdom In oviparous animals- e.g. frog- takes time to build up yolk and cytoplasm for embryo- 3years to sexual maturity

2. The cells from which sperm develop continue to be generated by mitosis through life of individual - not believed to happen in female. 3. Oogenesis marked by long resting periods whereas spermatogenesis is continuous. Sperm and egg are very specialised cells The egg is full of molecules needed for nutrition, metabolism and development of the embryo

Remember the analogy with “A store cupboard” The sperm cell is specialized to move to the egg and penetrate it. The head of the sperm is tipped with a special body the acrosome- contains enzymes to help the sperm penetrate the egg. The sperm cell contains a large number of mitochondria to provide the energy (ATP) needed to move the tail or flagellum for motility. The sperm cell contains little else

Remember the analogy with “a lean machine”

Fertilisation Brings male and females gametes together – produces diploid zygote It also activates the egg, triggering the beginning of embryonic

development We looked at the sea urchin as an example- one of the best studied model organisms (lecture 3) for fertilization. It is not a vertebrate but the process is similar in vertebrates. The fertilization occurs externally- in sea water. A jelly coat on the egg attracts the sperm The following are the steps involved in fertilization as worked out in the sea urchin. They are illustrated (numbered) on Fig 47.3 below 1. Acrosomal reaction: specialised vesicle at the tip of the sperm head (the acrosome) contains hydrolytic enzymes → digest jelly coat. 2. Acrosomal process- these are actin filaments that are stimluated to extend from the sperm head through the coat and bind to receptors on the vitteline membrane- “lock and key” recognition system to ensure correct species fertilises the egg. 3. •Contact and fusion of egg and sperm plasma membranes. •Depolarisation of the membrane to prevent polyspermy POLYSPERMY: fertilization by multiple sperm which would lead to an increased genetic compliment and in non-viable zygote 4. Entry of sperm nucleus 5. Cortical reaction- depolarisation of the membrane also leads to Ca2+ release from endoplasmic reticulum in a wave across the egg. Ca2+ brings about the fusion of numerous vesicles in cytoplasm with egg membrane releasing enzymes →swelling of perivitteline space →hardening of the vitteline layer →clipping of sperm-binding receptors →forming fertilisation envelope ULTIMATELY A LONG TERM BLOCK TO POLYSPERMY

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The Ca2+ release can be visualised in the egg →fluorescent dye that activates when binds Ca2+ Campbell and Reece Fig 47.4 Ca2+ release also causes egg activation- increasing rate of cellular respiration and protein synthesis. This implies that the entry of sperm causes egg activation but doesn’t supply any material needed- can be achieved artificially by ↑ Ca2+ ⇒activation can be induced The timeline of fertilisation in the sea urchin

Note in particular that within 2 seconds of sperm binding, the acrosomal reaction has occurred and the fast block to polyspermy is in place. Within 1 minute the cortical reaction has also occurred and the fertilisation envelope is in place giving the long term block to polyspermy. In the next 4 minutes or so the egg is activated. As metabolism is activated the sperm nucleus swells and after about 20 min the egg and sperm nuclei merge creating the diploid nucelus of the zygote. DNA synthesis begins and the first mitotic cell division occurs after only 90 mins. In other organisms- process is very similar - differences mainly in timing and in stage of maturation of egg (e.g. human stalled at metaphase of meiosis II) Fertilisation in mammals- differences

1. Fertilisation is internal 2. Egg cloaked in follicle cells released with egg 3. Sperm undergoes capacitation in uterus- molecular changes that enable sperm to

enter the egg 4. Egg has tough extracellular matrix called the zona pellucida - presents receptors

for sperm binding. 5. Binding of the receptor leads to acrosomal reaction- entry through zona pellucida 6. No known fast block to polyspermy but similar cortical reaction for slow block 7. Whole sperm taken into the egg- base used to form centrioles for spindle 8. Nuclei do not fuse but both nuclear envelopes disperse- chromosomes align on

spindle in cytoplasm. 9. First cell division slower- 12-36 hours

Campbell and Reece Fig 47.5 Key points in lecture 3

•Sexual reproduction ensures genetic variation and adaptability in the population

•In sexual reproduction two haploid gametes (n) produced by parents fuse to form a new unique diploid (2n) individual.

•In the production of gametes (sperm and ovum), meiosis (cell division) achieves a halving of the genetic material

•There are two phases in meiosis, meiosis I separates homologous chromosomes and meiosis II separates sister chromatids

•Spermatogenesis produces the male haploid gametes- sperm

•Oogenesis produces the female haploid gametes - ova/ eggs

•Sperm and ova are very specialised cell types and the differences between them are related to their specialised functions. This is the basis for differences between the

processes of spermatogenesis and oogenesis.

•Fertilisation serves two functions: it brings the haploid gametes together to form the new individual and it activates developmental processes in the egg

•The sea urchin presents a well studied example of fertilisation with many key features shared by all animals: The acrosomal reaction, the cortical reaction, block to polyspermy, and egg activation.

•Mammalian fetilisation differs in a number of aspects (e.g. timing, stage of egg on fertilisation, internal fertilisation) but includes all of the above features. Lecture 3: Learning outcomes: you should be able to…. A) Discuss the value of sexual reproduction. B) Describe how egg and sperm are generated by meiosis (you don’t have to draw the detailed diagrams but know the overall progression and terminology – especially differences between production of egg and sperm). C) Discuss the structure – function relationships between egg and sperm and how they relate to the generation of each during gametogenesis. D) Present the steps involved in fertilisation – particularly know what polyspermy is and the mechanisms used to prevent it. Key terms to be familiar with: gametes, sperm, spermatogenesis, spermatogonia, primary and secondary spermatocytes, spermatids, spermiogenesis, acrosome, ovum, oogenesis, oogonia, primary and secondary oocytes, polar body, fertilization, zygote, acrosomal reaction, acrosomal process, polyspermy, cortical reaction, egg activation, zona pellucida.