- 1 Individual Assessments
- 1.1 Lab 1 Assessment
- 1.2 Lab 2 Assessment
- 1.3 Lab 3 Assessment
- 1.4 Lab 4 Assessment
- 1.5 Lab 7 Assessment
- 2 Lab Attendance
Lab 1 Assessment
1. Identify the origin of In Vitro Fertilization and the 2010 nobel prize winner associated with this technique and add a correctly formatted link to the Nobel page.
The History of In Vitro Fertilisation
In vitro fertilisation (IVF) refers to the process of artificial fertilisation conducted ex vivo. The IVF technique was first described for non-human use. The earliest known research conducted was by Walter Heape from Cambridge University in the 1890s who reported the first known case of embryo transplantation in rabbits. In 1959, Dr. Min Chueh Chang published his work in Nature describing the first successful mammalian live birth (rabbits) after IVF therapy.
Eventually, the use of IVF for humans became a possibility and then a reality: in 1978, the first successful birth from IVF occurred in England. The success of this IVF birth is credited to Patrick Steptoe and Robert Edwards. In 2010, Edwards was awarded the Nobel Prize in Medicine for the development of human IVF therapy. Because of IVF, many couples have been given a chance to conceive. However, the history of IVF is still in the making with constant improvements in the technology being developed and applied.
2. Identify and add a PubMed reference link to a recent paper on fertilisation and describe its key findings (1-2 paragraphs).
Research in Fertilisation
In order for fusion between mammalian gametes to occur, a spermatozoon must first pass through the external layers surrounding the oocyte: the cumulus oophorus and the zona pellucida (ZP). It is believe that the acromosome reaction (AR) of the spermatozoa starts upon contact with the zona pellucida. Consequently, the cumulus cell layer is typically removed in studies of mouse sperm-oocyte interactions in order to facilitate fertilisation. The recent experiments of Jin et al.  sought to answer the question: "Where does the fertilising mouse spermatozoon begin the AR - in the cumulus [of the oocyte] or the zona pellucida?" Jin et al.  utilised fluorescence microscopy and transgenic mouse spermatozoa to conduct their investigation. Additionally, Jin et al.  used cumulus-free oocytes and cumulus-enclosed oocytes to study the role of the cumulus cells in fertilisation.
From the experiment, Jin et al.  found that most fertilising spermatozoa begin the AR before their first contact with the ZP. The significance of this finding was that the spermatozoa with intact acromosomes at the ZP seldom had the ability to penetrate through . In contrast, spermatozoa which had already began the AR could easily penetrate the ZP. In regards to the role of the cumulus cells, it was found that cumulus-enclosed oocytes had a higher incidence of fertilisation compared to cumulus-free oocytes . Moreover, cumulus-free oocytes had an increased incidence of in vitro fertilisation when incubated with other cumulus-enclosed cells; this finding suggests that cumulus cells harbour an important role in fertilisation . However, it is notable that when cumulus-free oocytes were incubated in a cumulus-conditioned medium, no increase in fertilisation rate was noted. Overall, two conclusions were made: firstly, that the AR is required by the spermatozoa prior to meeting the ZP for effective fertilisation. Secondly, the cumulus oophorus confers benefit in increasing the chance of fertilisation.
--Mark Hill 16:58, 11 September 2012 (EST) Question 1 is answered well and linked to appropriate resources. Question 2 is also quite a complete answer to what I requested. The formatting of your description could have been better organised, while it is correct to cite the paper when referring to the findings, this is a little overboard with 9 times within 2 paragraphs. If you had organised the information differently this could have been reduced to 1-2 citations within the text. Alternatively the findings could have been provided as a bullet or numbered list. 10/10
Lab 2 Assessment
1. Upload an image from a journal source relating to fertilization or the first 2 weeks of development as demonstrated in the practical class. Including in the image “Summary” window: An image name as a section heading, Any further description of what the image shows, A subsection labeled “Reference” and under this the original image source, appropriate reference and all copyright information and finally a template indicating that this is a student image.
Patterns of ZPC Deposition in Porcine Oocyte-Cumulus Complexes
Immunofluorescence Detection for ZPC and Ubiquitin in a Porcine Oocyte 
2. Identify a protein associated with the implantation process, including a brief description of the protein's role (1-2 paragraphs).
Trophinin and Implantation
Trophinin is a membrane protein expressed in chorionic villi trophoblasts and in the maternal endometrium. In the early stages of pregnancy, trophinin is strongly expressed along with tastin and bystin, which form a complex; this complex mediates apical cell adhesion between the trophoblasts and the endometrial epithelial cells. The time frame in which trophinin is expressed on the apical aspect of the endometrial cells coincides with the "implantation window"; the period in which successful implantation is possible. Trophonin-trophonin adhesion during implantation occurs via signal transduction with bystin and tastin. As a consequence of trophinin-trophinin adhesion, trophectoderm cells become activated for implantation. Moreover, there have been reports that endometrial epithelial cells undergo apoptosis upon blastocyst adhesion; human trophoectoderm cells express the Fas ligand which interacts with Fas expressed on the endometrium. However, other studies have shown that trophinin-mediated cell adhesion can induce endometrial cell apoptosis through mechanisms other than the Fas/FasL cascade.
In regards to ectopic pregnancies located within the fallopian tube, research has shown that trophinin is strongly expressed by both the embryonic trophoblasts and maternal fallopian tube epithelium, induced by human chorionic gonadotrophin (hCG). These findings highlight the function of trophonin in facilitating implantation in conjunction with its role in the pathogenesis of ectopic pregnancies.
--Mark Hill 17:04, 11 September 2012 (EST) Question 1 image has been correctly uploaded and contains all the requested information in the summary box. Question 2 is a good description of this recent paper on trophinin and Implantation. 10/10
Lab 3 Assessment
1. Identify the difference between "gestational age" and "post-fertilisation age" and explain why clinically "gestational age" is used in describing human development.
Gestational Age versus Post-Fertilisation Age
Gestation is the period of time between conception and birth (Kaneshiro, 2011; Vishton, 2011). Gestational age is the developmental age of the conceptus based on the presumed first day of the last normal menstrual period to the current date, measured in weeks (Kaneshiro, 2011; Vishton, 2011). In contrast, post-fertilisation age refers to the age of the conceptus expressed in elapsed time since fertilisation (Vishton, 2011). Gestational age is approximately two weeks greater than post-fertilization age (Kaneshiro, 2011; Vishton, 2011). Gestational age is used in human development because its start date can be determined by asking the mother when was the presumed first day of the last normal menstrual period (Kaneshiro, 2011; Vishton, 2011). In contrast, the moment of fertilization must be inferred (Vishton, 2011).
Kaneshiro, N. K. (2011). Gestational age. Retrieved from http://www.umm.edu/ency/article/002367.htm
Vishton, P. M. (2011). Embryo Foetus Development Stages. Retrieved from http://www.livestrong.com/article/92683-embryo-fetus-development-stages/
2.Identify using histological descriptions at least 3 different types of tissues formed from somites.
Tissues Derived From Somites
1. Bone (Sclerotome)
Bone tissue consists of cells separated by an extracellular matrix with organic and inorganic components (Marieb, Wilhem & Mallatt, 2010). The organic components of bone consists of cells, collagen fibres and ground substance. The cells include osteoprogenitor cells which give rise to osteoblasts: the producers of new bone matrix (osteoid). Mature osteoblasts, called osteocytes, are trapped in lacunae where they maintain the mature bone; osteocytes may revert to osteoblasts in the incidence of a fracture. Osteoclasts are multinucleated cells with ruffled plasma membrane borders and are involved in bone resorption. Bone resorption is important for bone remodelling to improve tensile strength as well as remodel the newly deposited woven bone into mature bone after a fracture. There are two types of mature (lamellar) bone: compact bone and spongy bone (Marieb et al., 2010). The compact bone occurs towards the periphery and is arranged in Haversian systems: lamellae concentrically arranged around a central Haversian canal containing blood vessels, nerves and osteocytes (Marieb et al., 2010). The different Haversian systems communicate with each other, the periosteum and endosteum through the Volkmann's canals. In contrast, spongy bone in the mature adult appears towards the centre of the diaphysis and metaphysis and is arranged in bony shelves (trabeculae) (Marieb et al., 2010). The gross porous arrangement of spongy bone is important for housing the bone marrow (Marieb et al., 2010)
2. The Skin (Dermotome)
a. Dermis: The dermis is made up of two main regions: (1) the superficial papillary layer and (2) the deeper reticular layer (Marieb et al., 2010). The superficial papillary layer makes up 20% of the dermis and is areolar connective tissue consisting of collagen and elastic fibres; it includes the dermal papillae which extend into the overlying epidermis to strengthen the dermal-epidermal junction and increase surface area for nutrient, gas and waste exchange with the avascular epidermis (Marieb et al., 2010). The reticular layer is composed of dense irregular connective tissue with thick bundles of collagen and elastic fibres arranged in different planes (Marieb et al., 2010). Other cells interspersed among the connective tissue of the dermis include fibroblasts, macrophages, mast cells and other white blood cells including lymphocytes(Marieb et al., 2010). The dermis is highly vascular and supplied with nerve fibres (Marieb et al., 2010). There are two vascular plexuses; the deep dermal plexus and the subpapillary plexus (Marieb et al., 2010). These vessels serve not only for nutrient supply to the dermis and epidermis, but for temperature regulation as well (Marieb et al., 2010).
b. Hypodermis: The subcutaneous layer, or fatty hypodermis, consists of areolar and adipose connective tissue(Marieb et al., 2010). The cellular components include adipocytes as well as white blood cells (Marieb et al., 2010). The hypodermis serves to store fat and anchor the skin to underlying structure in a manner that the skin can slide over structures(Marieb et al., 2010). Additionally, the adipose in the hypodermis serves an insulator.
3. Skeletal Muscle (Myotome) Skeletal muscle fibres come together to form a larger skeletal muscle surrounded by different levels of connective tissue "coats": the epimysium surrounds the whole skeletal muscle, the perimysium covers each fascicle and the loose CT endomysium separates each skeletal muscle fibre (Marieb et al., 2010). The skeletal muscle fibres are long cylindrical cells with a diameter between 10-100um (Marieb et al., 2010) . These muscle fbres are formed by the fusion of embryonic cells and hence contain many nuclei which are located at the periphery of each fibre beneath the sarcolemma, the skeletal muscle cell membrane (Marieb et al., 2010). These muscle fibres appear striated because of the internal organelles of the muscle fibres: myofibrils, the contractile organelles of muscle tissue (Marieb et al., 2010).
Marieb, E. N., Wilhelm, P. B., Mallatt, J. (2010). Human Anatomy (6th ed.). San Francisco, CA: Pearson Education, Inc.
--Mark Hill 17:10, 11 September 2012 (EST) Question 1 Clearly identified and cited the difference between "gestational age" and "post-fertilisation age". Question 2 you have also identified histological descriptions at least 3 different types of tissues formed from somites. For both questions you could have formatted the references and the reference list by using the <ref></ref> tags and simply inserted any text that you wanted in your list between the tags. 10/10
Lab 4 Assessment
Prenatal Diagnostic Techniques
1. Identify the 2 invasive prenatal diagnostic techniques related to the placenta and 2 abnormalities that can be identified with these techniques.
- Amniocentesis : Amniocentesis refers to sampling of the amniotic fluid by inserting a needle through the mother's anterior abdominal and uterine walls into the amniotic cavity by piercing the chorion and amnion. This technique is performed at 15 and 18 weeks gestation. Amniocentesis is often used to detect genetic disorders as it allows for chromosome analysis. One abnormality which can be detected is trisomy 21 (Down's Syndrome). Additionally, amniocentesis can be utilised for alpha-fetoprotein assays to detect neural tube defects like spina bifida .
- Chorionic Villus Sampling : Biopsies of trophoblastic tissue are obtained by inserting a needle through the mother's abdominal wall and uterine walls to the uterine cavity. Sampling can also be performed through the cervix with a polyethylene catheter, guided by real-time ultrasonography. CVS can be performed sooner than amniocentesis at 10 and 12 weeks of gestation. However, the rate of miscarriage from CVS is higher than amniocentesis. Like amniocentesis, CVS can be used to detect chromosomal abnormalities like trisomy 21 as well as trisomy 18. Additionally, Tay-Sachs disease can be detected with CVS .
The Therapeutic Use of Cord Stem Cells
2. Identify a paper that uses cord stem cells therapeutically and write a brief (2-3 paragraph) description of the paper's findings.
Fu et al. (2006) sought to isolate human umbilical cord mesenchymal stem cells (HUCMSCs) and transform them into dopaminergic neurons in vitro . The experiment was carried out in the interest of finding a potential cure for Parkinson's disease from HUCMSCs . The procedure involved isolating human mesenchymal stem cells from Wharton's jelly of the umbilical cord and culturing the HUCMSCs in a neuronal conditioned medium (NCM) . The differentiation of the HUCMSCs into dopaminergic neurons was induced through stepwise culturing with the NCM, sonic hedgehog and FGF-8 . The successfully transformed HUCMSCs into dopaminergic neurons were selected by positive immunohistochemistry staining for tyrosine hydroxylase (TH), the rate-limiting catecholaminergic synthesizing enzyme, and dopamine secretion . These neurons were then transplanted into the striatum of rats with induced Parkinson's disease by unilateral striatal lesioning with neurotoxin (6-hydroxydopamine hydrogen chloride). The effects of stem cell transplantation were examined in the Parkinsonian animals by quantification of rotations in the mice in response to amphetamine at 0, 1, 2, 3 and 4 months .
Despite a success rate of 12.7% of transformed HUCMSCs, the study found that the original number of HUCMSCs doubled after 3 days of culture . Additionally, the transformed HUCMSCs were still viable in the rats 4 months post-transplantation without the need for immunosuppression . These findings highlight HUCMSCs as a potentially safe source of organs due to their viability post-surgery and the lack of a negative host response to the newly transplanted tissues. Additionally, positive TH staining showed migration of the transformed HUCMSCs rostrally and caudally from the location of implantation . Hence, the transformed HUMSCs were able to integrate properly into the patient (Parkinsonian mouse), putting forth their suitability as a tissue source for transplantation.
The clinical effects of the HUCMSCs on the Parkinsonian mice were assessed by comparing test subjects to two control groups. The first control group consisted of normal, non-Parkinsonian mice with a low score of rotation in response to amphetamine. The second control group contained Parkinsonian mice which received no treatment and had a high rotation score . It was found that the second control group showed no improvement in rotation score and deteriorated further over time. Similarly, mice treated with non-transformed HUCMSCs did not have observable improvements and had a similar deteriorating rotation score to the untreated Parkinsonian animals . This finding highlights that untransformed HUCMSCs confer no clinical benefit in the setting of simulated Parkinson's disease . The Parkinsonian mice treated with transformed HUCMSCs at first had no observable improvement but over time showed significantly improved rotational scores relative to the Parkinsonian control group . However, the transformed HUCMSCs group did not show improvement to the extent of returning to a normal level (non-Parkinsonian rat). These findings suggest that HUCMSCs could be a potential stem cell source for transplantation, however, the procedures for HUMSCs transformation and transplantation must first be reviewed . Fu et al. (2006) suggested that, the number of dopaminergic neurons of implanted cells may have been relatively inadequate to alleviate the Parkinsonism symptoms in the affected rats. Additionally, the transplanted cells may take time to integrate into the host brain: only two rats in the transformed HUCMSCs group survived for at least 8 months, with amphetamine-induced rotation behavior remaining similar to that 4 months after transplantation. Hence, in order to properly assess HUCMSCs to treat Parkinson's disease, the long-term effects of transplantation must be studied .
- Moore, K. L., Persaud, T. V. N. & Torchia, M. G. (2013). The Developing Human (9th ed.). Philadelphia, PA: Elsevier Saunders.
--Mark Hill 17:18, 11 September 2012 (EST) Question 1 well answered in terms of technique and specific abnormalities. Question 2 human umbilical cord mesenchymal stem cells in Wharton's jelly have been used in several studies as potential sources of therapeutic cells. Perhaps a more recent paper next time. 10/10
Lab 7 Assessment
Muscle Satellite Cells
1. (a) Provide a one sentence definition of a muscle satellite cell
Satellite cells are quiescent cells located beneath the basal lamina of each myofibre and function as myogenic precursors (stem cells) for postnatal muscle growth and repair .
(b) In one paragraph, briefly discuss two examples of when satellite cells are activated ?
Muscle satellite cells are at rest (G0) when skeletal muscle is not active. Activation can occur in the settings of physical activity and mechanical trauma . When satellite cells are activated, they proliferate and are converted to myoblasts which further differentiate and fuse with existing muscle fibres or form new fibres . The myonuclei accumulated in the tissue are of key importance for myogenesis: they aid in increasing protein synthesis and enable muscle growth (hypertrophy and hyperplasia) and regeneration. Without satellite cells, mature muscle fibres are unable to undergo regeneration . There is much speculation as to the exact mechanisms and factors which activate muscle satellite cells. It is proposed that strenuous exercise, mechanical muscle injury or myodegenerative disease result in the inflammatory response which recruits neutrophils and macrophages to the area of damage; the inflammatory mediators released in conjunction with growth factors secreted by the myofibres promote myosatellite activation . One proposed "triggering agent" is the production of sphingosine-1-phosphate from the inner part of the plasma membrane leading to satellite cell entry into the cell cycle . Additionally, mechanical stretch to the muscle fibre has been shown to trigger intracellular signals, such as nitric oxide synthesis which results in hepatocyte growth factor (HGF) release . Nitric oxide also induces follistatin, a fusigenic secreted molecule, which is an antagonist to myostatin. Myostatin is expressed by quiescent satellite cells and exerts a negative effect on satellite cell activation . IGF-IEa and MGF have also been implicated in satellite cell activation: Hill et al. (2003) demonstrated that these mediators are produced by active muscle in rodents and appear to be positive regulators of muscle hypertrophy. However, whilst MGF is acutely induced and is said to precede satellite cell activation, IGF-IEa has a delayed effect involved in the later phase of regeneration. Interestingly, in dystrophic muscles MGF is not produced, suggesting the importance of effective muscle repair to avoid the pathogenesis of muscular dystrophy syndromes .
Once activated, the satellite cells migrate out of the basal lamina and enter the cell cycle with coexpression of Pax7 and MyoD . This process occurs in conjunction with the Notch signaling pathway. The skeletal myoblasts that are produced divide, express myogenin and downregulate Pax7 then fuse to form myofibres.
The Effects of Motor Nerve Damage on Skeletal Muscle
2. In one brief paragraph, describe what happens to skeletal muscle fibre type and size when the innervating motor nerve sustains long term damage such as in spinal cord injury?
Chronic spinal cord injury (SCI) affects muscles below the level of the lesion. In terms of fibre type, one review describes the progressive change in fibre type toward faster phenotypes. The fibre phenotype is classified according to its myosin heavy chain (MHC) molecule which is an actin-based motor protein associated with muscle fibre contraction. These three MHC isoforms are MHC I, MHC IIa, and MHC IIx . In SCI there is a reduction of type I fibres (slow) and upregulation of type IIA and IIX fibres (fast). The metabolism of the fast fibres is characterised by a predominantly anaerobic metabolism with fewer mitochondria, unlike type I fibres which undergo aerobic metabolism . This is in part due to a reduction in absolute activities of the muscle metabolic enzymes in SCI, favouring the fast glycolytic/oxidative type which fatigue more easily .
In terms of fibre size, a study by Castro et al. (1998)  found that all fibre types underwent significant atrophy and decreased in size from the 6th to 24th week after injury. Moreover, average fibre cross-sectional area decreased by 22% by the 6th week post-injury . The changes were accompanied by either complete paralysis or loss of force with increased susceptibility to fatigue depending on the extent of injury . Note that whilst type II fibre atrophy is seen during the first months after complete SCI, type I fibres undergo atrophy in the later stages .
Interestingly, the level of the motor neuron lesion can affect the outcome of the muscle atrophy: a study by Stilwill and Sagha found that lower motor neuron lesions led to muscle fibre grouped atrophy and fibre-type grouping, whereas upper motor neuron lesions led to preferential atrophy of type II fibers with fibre-type grouping.
Lab 1 --Z3333038 11:49, 25 July 2012 (EST)
Lab 2 --Z3333038 10:05, 1 August 2012 (EST)
Lab 3 --Z3333038 10:01, 8 August 2012 (EST)
Lab 4 --Z3333038 10:00, 15 August 2012 (EST)
Lab 5 --Z3333038 09:59, 22 August 2012 (EST)
Lab 6 --Z3333038 10:10, 29 August 2012 (EST)
Lab 7 --Z3333038 10:09, 12 September 2012 (EST)