11.4 Sexual reproduction Flashcards

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1
Q

What is sexual reproduction?

A
  • A form of reproduction that involves the fusion of two haploid gametes.
  • The production of these gametes occurs during gametogenesis, which is known as spermatogenesis and oogenesis in males and females, respectively.
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2
Q

Define gametogenesis

A

The process by which cells of the germinal epithelium undergo cell division and differentiation to form haploid gametes.

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3
Q

What is spermatogenesis?

A

The production of sperm (takes place in the testes).

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4
Q

Where does the production of sperm take place?

A

In the testes

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5
Q

What are testes composed of?

A

Seminiferous tubules with interstitial cells (sometimes called Leydig cells) filling up the gaps in between.

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6
Q

Cross section of seminiferous tubules and interstitial cells in the testes

A
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7
Q

What is the outer layer of the seminiferous tubule made up of?

A

A layer of cells called the germinal epithelium.

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8
Q

What is the function of the stem cells of the germinal epithelium?

A
  • These cells, also known as spermatogonia (these are diploid cells, 2n), divide by mitosis to produce more spermatogonia.
  • Out of the two daughter cells produced, one renews the stock of spermatogonia in the germinal epithelium, while the other will be involved in sperm production.
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9
Q

Step 1 of how spermatogonia develop into sperm

A
  • Spermatogonia grow into larger cells called primary spermatocytes (2n).
  • Each primary spermatocyte carries out the first division of meiosis to produce two secondary spermatocytes (n).
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10
Q

Step 2 of how spermatogonia develop into sperm

A

Secondary spermatocytes carry out the second division of meiosis to produce two spermatids (n).

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11
Q

Step 3 of how spermatogonia develop into sperm

A
  • Spermatids become associated with nurse cells, called Sertoli cells, which help the spermatids to develop into spermatozoa (n) or sperm.
  • This is an example of cell differentiation.
  • Sertoli cells provide nurture and support.
  • The function of Sertoli cells is to nourish the developing sperm cells through the stages of spermatogenesis.
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12
Q

Step 4 of how spermatogonia develop into sperm

A

Sperm detach from Sertoli cells and are eventually carried out of the testis by the fluid in the center of the seminiferous tubule.

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13
Q

All steps of how spermatogonia develop into sperm

A
  • Spermatogonia grow into larger cells called primary spermatocytes (2n). Each primary spermatocyte carries out the first division of meiosis to produce two secondary spermatocytes (n).
  • Secondary spermatocytes carry out the second division of meiosis to produce two spermatids (n).
  • Spermatids become associated with nurse cells, called Sertoli cells, which help the spermatids to develop into spermatozoa (n) or sperm. This is an example of cell differentiation. Sertoli cells provide nurture and support. The function of Sertoli cells is to nourish the developing sperm cells through the stages of spermatogenesis.
  • Sperm detach from Sertoli cells and are eventually carried out of the testis by the fluid in the center of the seminiferous tubule.
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14
Q

Diagram showing the stages of spermatogenesis

A
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15
Q

What is the function of Leydig cells?

A

Leydig cells in the interstitial space of the testis produce the hormone testosterone, which allows spermatocytes to complete the meiotic divisions and mature into spermatozoa.

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16
Q

What are spermatogenesis and male fertility dependent upon?

A

The presence of testosterone in the testis.

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17
Q

Where does the development of sperm cells occur?

A

From the outside to the center (of the seminiferous tubule), with the cells in the center being the most mature.

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18
Q

Diagram os the stages of spermatogenesis in the wall of the seminiferous tubule

A
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19
Q

What is oogenesis?

A

This involves the production of ova (egg cells), which takes place in the ovaries of a female.

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20
Q

How many primary follicles is a female born with?

A

Around 400,000

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21
Q

What does the primary follicle consist of?

A

A primary oocyte, surrounded by a single layer of follicle cells.

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22
Q

When is the development of the primary oocyte halted?

A

At the first meiotic division.

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23
Q

Picture (micrograph) of a primary follicle

A
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24
Q

Stage 1 of oogenesis

A
  • At puberty, FSH (follicle stimulating hormone) begins to be produced and causes some follicles to develop each month.
  • This allows the oocyte to complete the first meiotic division.
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25
Q

Stage 2 of oogenesis

A
  • The first meiotic division forms two cells.
  • The smaller of the two cells is the polar body, which eventually degenerates.
  • The larger cell becomes a secondary oocyte, which enters meiosis II.
  • Meiosis II is halted at metaphase II if fertilization does not occur.
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26
Q

Stage 3 of oogenesis

A

If fertilisation does occur, meiosis II continues and a second polar body and a ovum are produced.

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27
Q

All steps of oogenesis

A
  • The process begins during fetal development. A female is born with around 400, 000 primary follicles. The primary follicle consists of a primary oocyte, surrounded by a single layer of follicle cells.
  • These cells (oogonia) undergo cell growth until they are large enough to undergo meiosis (becoming primary oocytes)
  • The primary oocytes begin meiosis but are arrested in prophase I
  • The primary oocytes remain arrested in prophase I until puberty, when a girl begins her menstrual cycle
  • Each month, hormones (FSH) will trigger the continued division of some of the primary oocytes
  • These cells will complete the first meiotic division to form two cells of unequal size
  • One cell retains the entirety of the cytoplasm to form a secondary oocyte, while the other cell forms a polar body
  • The polar body remains trapped within the follicle until it eventually degenerates
  • The secondary oocyte begins the second meiotic division but is arrested in metaphase II
  • The secondary oocyte is released from the ovary (ovulation) and enters into the oviduct (or fallopian tube)
  • The follicular cells surrounding the oocyte form a corona radiata and function to nourish the secondary oocyte
  • If the oocyte is fertilized by a sperm, chemical changes will trigger the completion of meiosis II and the formation of another polar body (the first polar body may also undergo a second division to form a third polar body)
  • Once meiosis II is complete, the mature egg forms an ovum, before fusing its nucleus with the sperm nucleus to form a zygote
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28
Q

Why are differentiation and maturation in the case of oogenesis longer processes than in spermatogenesis?

A
  • Because of the build-up of a store of cytoplasmic enzymes, mRNAs, organelles, and metabolic substrates in the cytoplasm of the oocyte.
  • This happens every month after puberty when the primary follicle starts to mature.
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29
Q

Similarities between spermatogenesis and oogenesis

A

They both involve mitosis, cell growth, two divisions of meiosis, and differentiation and result in (n) gametes.

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30
Q

Table showing similarities and differences between spermatogenesis and oogenesis

A
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31
Q

Diagram showing the stages of oogenesis

A
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32
Q

Where do human sperm cells develop?

A

Seminiferous tubules (testes)

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33
Q

When/where does oogenesis begin?

A

In the womb

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34
Q

What is the role of testosterone in spermatogenesis?

A

It stimulates the meiotic divisions of spermatogonia into spermatozoa.

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35
Q

Annotating diagrams of a seminiferous tubule and an ovary for the exam

A

You need to be able to annotate a diagram of a seminiferous tubule and an ovary.

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36
Q

Diagram of spermatogenesis

You only need to be able to annotate the seminiferous tubule in the middle.

A
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37
Q

Diagram of oogenesis

A
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38
Q

What are the different types of follicles?

A
  • As the primary follicle develops several layers of granulosa cells, it becomes known as the secondary follicle; and when it develops fluid-filled cavities, it is called a tertiary follicle.
  • The Graafian follicle is an advanced tertiary follicle where all the smaller fluid-filled cavities have fused together (forming one main cavity).
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39
Q

Which of the cells labeled in the testis diagram provide nourishment for developing sperm cells?

A

iii

This indicates a Sertoli cell which nourishes developing spermatozoa.

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40
Q

On the diagram below, which label represents a spermatogonium cell?

A

A

A is the spermatogonium because they are closest to the basement membrane.

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41
Q

Which label shows the ovulated ovum?

A

E

This is the ovulated ovum as you can see it has been released from the mature follicle within the ovary. The other labels are: A = Primary follicles; B = Oocyte; C= Zona Pellucida; D = Secondary Oocyte.

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42
Q

Annotating diagrams of a sperm and egg cell for the exam

A

You need to be able to annotate diagrams of a sperm cell and an egg cell.

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43
Q

Diagram of a sperm cell

A
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44
Q

Diagram of an ovum (egg cell)

A
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45
Q

Give the name of structure X.

A

Acrosome

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46
Q

What happens after copulation?

A
  • The sperm deposited in the female’s body start swimming toward the egg to fertilize it.
  • It is believed that the sperm follow a concentration gradient of chemicals secreted by the oocyte.
  • Once the sperm reach the egg, fertilization may occur.
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47
Q

What is fertilization?

A

The fusing of two gametes: an egg cell and a sperm cell.

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48
Q

Where does fertilization usually take place?

A

In the fallopian tube (also called an oviduct) in the female body.

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49
Q

Both the egg and sperm cells are ___, so the resulting zygote is ___.

A

Haploid

Diploid

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50
Q

What would happen if more than one sperm could fertilize an egg?

A
  • It would result in polyploid offspring, which is normally not desirable (this is called polyspermy).
  • There are mechanisms in place to prevent that from happening.
51
Q

Diagram showing the five steps of fertilization

A
52
Q

Step 1 of fertilization

A

A sperm cell penetrates the follicle cells and binds to the receptors of the zona pellucida.

53
Q

Step 2 of fertilization

A
  • The acrosomal reaction occurs: hydrolytic enzymes are released from the acrosome by exocytosis.
  • These enzymes make a hole in the zona pellucida, allowing the sperm to make its way to the plasma membrane of the egg.
54
Q

Step 3 of fertilization

A

There is contact between the sperm and egg, with the fusion of the sperm and egg plasma membranes.

55
Q

Step 4 of fertilization

A
  • The sperm nucleus enters the egg cytoplasm.
  • This triggers a number of metabolic and physical changes in the egg, that are collectively called egg activation.
  • The main effects are the completion of the second meiotic division and a rise in the intracellular concentration of calcium (a step that prepares for the cortical reaction).
56
Q

Step 5 of fertilization

A
  • The cortical reaction hardens the zona pellucida, preventing polyspermy.
  • This reaction involves the release of a mixture of enzymes, including several proteases, from cortical granules by exocytosis.
  • These enzymes diffuse into the zona pellucida and alter its structure by inducing hardening of the layer, as well as the destruction of sperm receptors.
  • As a result, additional sperm cannot bind to the zona pellucida or digest it to reach the oocyte.
57
Q

All steps of fertilization

A
  1. A sperm cell penetrates the follicle cells and binds to the receptors of the zona pellucida.
  2. The acrosomal reaction occurs: hydrolytic enzymes are released from the acrosome (a huge modified lysosome) by exocytosis. These enzymes make a hole in the zona pellucida allowing the sperm to make its way to the plasma membrane of the egg.
  3. There is contact between the sperm and egg, with the fusion of the sperm and egg plasma membranes.
  4. The sperm nucleus enters the egg cytoplasm. This triggers a number of metabolic and physical changes in the egg, which are collectively called egg activation. The main effects are the completion of the second meiotic division and a rise in the intracellular concentration of calcium (a step that prepares for the cortical reaction).
  5. The cortical reaction hardens the zona pellucida, preventing polyspermy. This reaction involves releasing a mixture of enzymes, including several proteases, from cortical granules by exocytosis. These enzymes diffuse into the zona pellucida and alter its structure by inducing the layer’s hardening and destroying sperm receptors. As a result, additional sperm cannot bind to the zona pellucida or digest it to reach the oocyte.
58
Q

What is the importance of the cortical reaction?

A

It represents a major block to polyspermy in most mammals.

59
Q

When you describe the fertilization process, ensure that you have mentioned ___

A
  • The acrosome reaction.
  • The fusion of the plasma membrane of the egg and sperm.
  • The cortical reaction.
60
Q

Colored scanning electron micrograph showing a sperm about to enter the egg.

A
61
Q

What are the two different forms that fertilization can be in?

A

Internal or external

62
Q

In what conditions is fertilization often external?

A

In marine or freshwater environments.

63
Q

What is the difference between external and internal fertilization?

A
  • External fertilization is a method of fertilization in which two haploid gametes, a sperm, and an egg cell, fuse together outside of the parents’ body.
  • In contrast, internal fertilization involves the transfer of sperm into the female’s body for fertilization to occur.
64
Q

When does external fertilization work best?

A
  • When vast quantities of eggs and sperm are produced to increase the chances of fertilization.
  • This is needed because the eggs and sperm can be predated upon by other animals.
  • Other environmental factors, such as temperature or pH, may also influence the success rates of fertilization.
65
Q

Courtship rituals- external fertilization

A

Courtship rituals make sure that the male and female sex cells are released within a short distance of each other, but even so, the chances of fertilisation are very low.

66
Q

What animals often use internal fertilization and why?

A
  • Terrestrial animals often use internal fertilization as sperm need fluid in which to swim to the egg.
  • In this case, the chances of fertilization are much greater since the sex cells are closer together when released.
  • This internal approach involves fewer eggs and sperm, but the proximity between the two increases the success rate.
67
Q

What is the role of the acrosome in sperm?

A

It contains enzymes to facilitate the penetration of the egg.

68
Q

During fertilization in humans, where do the acrosome and the cortical reactions occur most often?

A

Acrosome reaction: fallopian tubes

Cortical reaction: fallopian tubes

Since fertilization occurs in the Fallopian tube, that is where both the acrosome and cortical reactions occur.

69
Q

What happens following fertilization?

A
  • The ovum divides by mitosis, and after approximately 48 hours there is a four-cell embryo.
  • At this stage, the embryo is still in the oviduct, where fertilization took place.
  • The embryo slowly divides further and, at the same time, migrates down the oviduct to the uterus.
  • After approximately seven days, the embryo reaches the uterus.
70
Q

Diagram of the journey from a fertilized egg to a blastocyst (from the oviduct to the uterus)

A
71
Q

What is the embryo called after it reaches the uterus?

A

A blastocyst (it has around 125 cells)

72
Q

Why are there differences among the cells of the blastocyst?

A

Due to unequal division of cytoplasm during cytokinesis of some divisions, there are differences among the cells of the blastocyst.

73
Q

Describe the structure of the blastocyst

A
  • It is made up of a thin-walled hollow structure that contains an outer layer of cells and a cluster of cells called the inner cell mass.
  • This inner cell mass gives rise to the embryo, while the outer cell layer develops into the placenta and other supporting tissues needed for fetal development within the uterus.
74
Q

Diagram of the ‘hollow ball’ structure of the human blastocyst

A
75
Q

What is the next stage in pregnancy after the formation of the blastocyst?

A
  • The next stage in pregnancy is the implantation of the blastocyst in the endometrium (lining) of the uterus.
  • The egg cell contains reserves needed for the early embryo to grow.
  • Once these are used up, the zona pellucida breaks down and allows the blastocyst to implant into the uterine wall.
  • Finger-like projections grow into the uterine wall and will develop into the placenta, allowing the embryo to access external supplies of the required nutrients.
76
Q

Why is the implantation of the blastocyst in the endometrium essential for the continuation of pregnancy?

A

Because the uterine lining provides the developing embryo with necessary nutrients and oxygen.

77
Q

Why is the endometrium not shed during pregnancy?

A
  • Normally, the endometrium is shed at the end of the menstrual cycle, but if the egg is fertilized, it is maintained throughout pregnancy.
  • This is achieved by the continued production of progesterone and estrogen.
78
Q

What is the role of hCG in pregnancy?

A
  • Early in pregnancy, the embryo (more specifically the outermost layer of cells that will develop into the placenta) starts to produce human chorionic gonadotropin (hCG), which stimulates the corpus luteum to continue the production of progesterone and estrogen, hormones that are essential for the maintenance of the endometrium.
  • Actually, hCG ensures that the corpus luteum remains until the placenta is fully established and can take over the role of progesterone and estrogen secretion.
79
Q

What is responsible for miscarriages?

A
  • The loss of the corpus luteum before the placenta is fully established.
  • After around the third month of pregnancy, the placenta takes over the role of progesterone and estrogen secretion to maintain the uterine lining throughout pregnancy.
80
Q

Which embryo-produced hormone maintains progesterone and estrogen secretion by the corpus luteum?

A

Human chorionic gonadotropin (hCG)

81
Q

Following fertilization, how does the zygote develop into a blastocyst?

A

Through mitotic division

82
Q

What is the placenta?

A
  • An organ that facilitates the exchange of material between the fetus and its mother.
  • Not all animals with internal fertilization have a fetus that develops inside its mother.
  • Birds, amphibians, and reptiles usually lay eggs.
83
Q

What are the two mammals that are not placental?

A
  • The echidna and the platypus, both belong to the order Monotremata.
  • They lay eggs but still suckle their young.
84
Q

Describe the structure of the placenta

A
  • The placenta is fetal in origin.
  • The fingerlike projections (chorionic villi) are villi that originate in the chorion.
  • These villi provide a maximum surface area for contact with the maternal blood.
  • The villi project into the intervillous space. Maternal blood collects in these spaces (maternal blood pools in Figure 2 ).
  • The fetal blood circulates in capillaries which lie very close to the surface of the villus, with only a few micrometers separating them from the maternal blood pools.
  • This short distance facilitates diffusion between the maternal blood and the fetal blood.
85
Q

Diagram showing human placenta

A
86
Q

What are the two important functions of the placenta throughout pregnancy?

A
  • Exchange of material to keep the fetus alive during pregnancy.
  • Production of progesterone and estrogen.
87
Q

Explain how the placenta is involved in the production of progesterone and estrogen.

A
  • At the beginning of the pregnancy, this task is performed by the corpus luteum.
  • However, the activity of the corpus luteum progressively decreases from the beginning of the eighth week.
  • Its role is entirely replaced by the placenta at the end of the first trimester (about 12 weeks).
88
Q

What materials pass from the mother to the fetus?

A
  • Oxygen
  • Glucose, amino acids, vitamins, minerals
  • Water
  • Hormones
  • Drugs, alcohol and other harmful substances
  • Some viruses (rubella, HIV)
89
Q

How does oxygen pass through the mother to the fetus?

A

Diffusion

90
Q

How do glucose, amino acids, vitamins, and minerals pass from the mother to the fetus?

A

Facilitated diffusion

91
Q

How does water pass from the mother to the fetus?

A

Osmosis

92
Q

How do hormones pass from the mother to the fetus?

A

Endocytosis

93
Q

How do drugs, alcohol, and other harmful substances pass from the mother to the fetus?

A

Diffusion

94
Q

How do some viruses (rubella, and HIV) pass from the mother to the fetus?

A

Via receptors

95
Q

What passes from the fetus to the mother?

A
  • Carbon dioxide
  • Urea
  • Water
  • Hormones (e.g. hCG)
96
Q

How does carbon dioxide pass from the fetus to the mother?

A

Diffusion

97
Q

How does urea pass from the fetus to the mother?

A

Facilitated diffusion

98
Q

How does water pass from the fetus to the mother?

A

Osmosis

99
Q

How do hormones (e.g. hCG) pass from the fetus to the mother?

A

Exocytosis

100
Q

What are the functions of the two fetal arteries and the fetal vein and how is this different in a newborn baby?

A
  • The two fetal arteries (or umbilical arteries) bring deoxygenated blood from the fetus to the placenta, while the fetal (or umbilical) vein (there is only one vein) brings oxygenated blood back from the placenta to the fetus.
  • This is the opposite of the situation in a newborn baby, where, of course, the lungs are functioning, and the arteries bring oxygenated blood to the tissues.
101
Q

What do all fetal venules and arterioles unite together to form?

A
  • All fetal venules unite together to form the fetal vein, while all fetal arterioles fuse to form the fetal arteries.
  • Both of these vessels lie in the umbilical cord which connects the fetus to the uterine wall.
102
Q

What does the fetal vein carry?

A

Oxygenated fetal blood away from the placenta.

Fetal arteries bring deoxygenated blood from the fetus to the placenta, while the fetal veins bring oxygenated blood back from the placenta to the fetus.

103
Q

Which hormones are secreted by the placenta?

A

Estrogen and progesterone

104
Q

The umbilical arteries carry blood from the fetus to the placenta and the umbilical vein carries blood from the placenta to the fetus.

How does the composition of the blood in the umbilical artery and the umbilical vein differ?

A

Blood in the umbilical arteries contains less glucose than blood in the umbilical vein.

105
Q

What is the gestation period?

A

The time that the embryo develops within the female’s body

106
Q

Describe gestation periods in different animals

A
  • It starts with fertilization and ends at birth.
  • It varies according to the species and correlates with the average body mass of the animals.
107
Q

Table showing gestation times and the average mass of the mother

A
108
Q

Describe the relationship between the average mass of a mother and the gestation period

A
  • Above a certain mass, there is a linear relationship between the average mass and the gestation period.
  • (The values are presented on a logarithmic scale because they cover such a wide range.)
  • The higher the average mass of an animal, the longer the gestation period.
  • Smaller mammals often have offspring that are helpless and need a lot of protection.
  • Larger mammals, such as bison, cows and elephants have young that are relatively well-developed.
  • This is directly linked to longer gestation periods.
109
Q

Graph showing mass versus gestation time

A
110
Q

Explain the hormonal controls of birth

A
  • After 9 months, the baby is fully grown and stretches the walls of the uterus – placing a strain on both the mother and infant
  • This strain induces the release of chemicals that trigger a rise in the levels of estrogen
  • Estrogen prepares the uterus for hormonal stimulation by increasing its sensitivity to oxytocin
  • Estrogen also inhibits progesterone, which was preventing uterine contractions from occurring while the fetus developed
  • Now that the uterus is primed for childbirth, the brain triggers the release of oxytocin
  • Oxytocin stimulates the uterine muscles to contract, initiating the birthing process (it also inhibits progesterone secretion)
  • The fetus responds to this uterine contraction by releasing prostaglandins, which triggers further uterine contractions
  • As the uterine contractions trigger the release of chemicals that cause further contractions, a positive feedback loop ensues
  • Contractions will stop when labor is complete and the baby is birthed (no more stretching of the uterine wall)
111
Q

Diagram showing the actions and effects of the hormones involved in birth (relative hormone concentrations during and after pregnancy)

A
112
Q

Explain how labor occurs

A
  • The endometrium secretes prostaglandins that start the uterine contractions.
  • Oxytocin stimulates the uterine muscles (myometrium) to contract.
  • It is the strongest stimulator of uterine contractions.
  • Oxytocin is controlled by positive feedback.
  • This means that, initially, the contractions are mild, but become stronger as more oxytocin is released.
  • This is called labour.
113
Q

What is birth?

A
  • The strength of contractions increases until the baby (along with the umbilical cord and placenta) is expelled from the uterus.
  • This is birth.
114
Q

Birth is mediated by positive feedback involving ___

A

Estrogen and oxytocin.

115
Q

Explain what happens during birth

A
  • As the uterus contracts, the baby is pushed down towards the cervix.
  • The pressure from the baby’s head helps the cervix to open (called dilation) and the amniotic sac breaks (waters breaking).
  • Contractions may continue for hours until the baby is pushed through the cervix and the vagina.
116
Q

How is milk production stimulated after birth?

A

After the baby is born, prolactin, a hormone produced by the anterior pituitary gland, stimulates milk production (also called lactation).

117
Q

Graph showing the relative hormone concentrations during and after pregnancy

A
118
Q

Why is there variation in the secretion of prolactin after birth?

A

After birth, prolactin production is stimulated when the baby suckles.

119
Q

A woman, in the final week of her pregnancy, is given an injection of oxytocin.

What would be the likely result?

A

She undergoes vigorous contractions of her uterine muscles.

120
Q

What are the roles of oxytocin and progesterone during childbirth?

A

Oxytocin: Causes uterine contractions

Progesterone: Level falls, causing oxytocin production

121
Q

When was synthetic estrogen (ethinyl estradiol) first synthesized and when was it introduced as a contraceptive?

A

It was first synthesised in 1938 and introduced as a contraceptive in 1943.

122
Q

What is estrogen pollution and what has caused it?

A
  • The widespread use of contraceptives, such as estradiol, has increased levels of these synthetic hormones in the environment.
  • This is sometimes referred to as estrogen pollution.
123
Q

What are the possible consequences of higher than normal levels of this hormone in the environment?

A
  • One possible outcome could be a lowering of the average sperm count in males.
  • A number of studies have concluded that the average male sperm count has declined by 50% over the last 50 years.
  • Other studies have suggested that the hormones were causing certain fish to show female characteristics.
124
Q

Effects of higher levels of estrogen in the environment- implications for governments

A
  • Both of these studies have shown correlations, but not a causal relationship.
  • Governments may legislate on the basis of these studies, but pharmaceutical industries, as well as the water waste-management industry, have strongly protested against any new laws because it would increase their costs.
  • Scientists use the correlation as a starting point for further studies.
  • A causal relationship should be demonstrated before any legislation can be considered or defended.
  • Other compounds comparable to estrogen are used intensively in agriculture.
  • Furthermore, other compounds, such as BPA ( Bisphenol A , used in bottles), are also widely used and have been shown to be endocrine disruptors.