User:Z3333865

Lab Attendance

Lab 1 --Z3333865 11:00, 25 July 2012 (EST)

Lab 2 --Z3333865 10:04, 1 August 2012 (EST)

Lab 3 --Z3333865 11:58, 8 August 2012 (EST)

Lab 4 --Z3333865 11:43, 15 August 2012 (EST)

Lab Exercises

Lab 1

Question 1

As stated by IVF-worldwide, the history of In Vitro Fertilization (IVF) and embryo transfer (ET) dates back as early as the 1890s. Walter Heape, a professor and physician at the University of Cambridge, England, had been conducting research on reproduction in a number of animal species. He reported the first known case of embryo transplantation in rabbits, long before the applications to human fertility were even suggested. IVF history

IVF-worldwide also explains that in 1965, Robert Edwards together with Georgeanna and Howard Jones at Johns Hopkins Hospital in the USA attempted to fertilize human oocytes in vitro. The first IVF pregnancy was reported in 1973 by the Monash research team of Professors Carl Wood and John Leeton in Melbourne, Australia. Unfortunately, this resulted in early miscarriage. The first ever IVF birth occurred in Oldham, England on July 25, 1978. This birth was the result of the collaborative work of Patrick Steptoe and Robert Edwards. IVF history

Robert Edwards was awarded the 2010 Nobel Prize in Physiology or Medicine for the development of human In Vitro Fertilization (IVF) therapy. His achievements have made it possible to help treat infertility, which affects more than 1 in 10 couples worldwide. 2010 Nobel Prize

Question 2

<pubmed>PMC3353509</pubmed>

Traditional IVF methods involve the assisted fertilization of the oocytes with the spermatozoa. This is performed in the laboratory, whereby the physiological conditions to which the gametes are normally exposed in vivo are simulated. However, INVO (intravaginal culture of oocytes), is a simplified procedure and alternative option to conventional IVF. This assisted reproduction procedure uses the maternal vaginal cavity for incubation, instead of laboratory equipment.

Investigated in this study is the outcome of the INVO procedure and how this compares to the conventional IVF methods. Data was obtained regarding pregnancy, live birth, and single live birth rates. Results of this study showed that the INVO procedure had comparable successful rates with traditional IVF.

Statistics from 2008 on traditional IVF: the pregnancy, live birth, and singleton live birth rates per oocyte retrieval were 41.6%, 33.8%, and 23%, respectively.

Statistics from this study on INVO: the pregnancy, live birth, and singleton live birth rates per oocyte retrieval were 40%, 31.2%, and 24%, respectively.

The study also concluded that the most significant factor determining the success rate was the age of the mother. In terms of pregnancy, live birth, and single live birth rates, a significant decrease was observed across the groups of age from ≤29 until ≥40 years old. Results obtained by this study suggest that INVO procedures could be a viable alternative treatment for infertile patients.

Link to paper on INVO

Lab 2

In-class exercise

Confirmation of homologous recombination and c-MYC2 expression in ES cell clones.

Confirmation of homologous recombination and c-MYC2 expression in ES cell clones.

(A) Genomic DNA of ES cell clones 1, 14, 18 and 19 and of wildtype ES cells (wt Bruce 4) was digested with EcoRI. Digested DNA was analyzed by Southern blotting with a 5′ probe and a 3′ probe. (wt) DNA fragment of the wildtype c-Myc locus; (rec.) DNA-fragment of recombined hc-Myc locus. (B) Protein extracts were prepared of ES cell clones 1, 14, 18 and 19 as well as of wildtype ES cells (wt Bruce 4) and of a human lymphoblastoid cell line (LCL 1.11). Human c-MYC2 (hu. c-MYC, ca. 62 kDa) was detected with antibody clone Y69. In wildtype ES cells murine c-MYC2 (mu. c-MYC, ca. 64 kDa) was detected. For loading control an antibody specific for glyceraldehyde-3-phosphat-dehydrogenase (GAPDH; ca. 36 kDa) was used. Western blot results were reproduced five times.

Lehmann FM, Feicht S, Helm F, Maurberger A, Ladinig C, et al. (2012) Humanized c-Myc Mouse. PLoS ONE 7(7): e42021. doi:10.1371/journal.pone.0042021

Copyright: © 2012 Lehmann et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Assessment task

Jam2 expression in mouse uterus during early pregnancy.

Question 1

Jam2 expression in mouse uterus during early pregnancy.

(A) In situ hybridization of Jam2 mRNA. (B) Real-time RT-PCR quantification of Jam2 mRNA. (C) JAM2 immunostaining. D1, day 1; D2, day 2; D3, day 3; D4, day 4; D4.5-I, implantation site at day 4 midnight; D4.5-NI, inter-implantation site at day 4 midnight; D5-I, implantation site on day 5; D5-NI, inter-implantation site on day 5; PD3, day 3 of pseudopregnancy; PD4, day 4 of pseudopregnancy; Arrow, embryo. Bar = 150 µm.

Su R-W, Jia B, Ni H, Lei W, Yue S-L, et al. (2012) Junctional Adhesion Molecule 2 Mediates the Interaction between Hatched Blastocyst and Luminal Epithelium: Induction by Progesterone and LIF. PLoS ONE 7(4): e34325. doi:10.1371/journal.pone.0034325

Copyright: © 2012 Su et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Question 2

A protein associated with the implantation process is Hand2.

It is known that levels of this protein increase in uterine cells as progesterone levels rise. In a more recent NIH funded study, researchers discovered that Hand2 is also the switch that turns off estrogen’s stimulating effect on the epithelium.

For the study, the researchers developed a laboratory strain of mice in which the uterus fails to make Hand2. It was found that exposure to progesterone halted growth of the uterine epithelium in mice with functioning genes for Hand2. However, despite exposure to progesterone, epithelial growth continued unchecked in the mice without Hand2 genes.

Furthermore, at the time of implantation, Hand2 was expressed in uterine cells that lie beneath the surface layer of epithelial cells. Experiments have shown that estrogen stimulates the production of growth factors, which cause cells in the epithelial layer to multiply and grow. When progesterone is produced, it spurs the release of Hand2, which stops the production of growth factors. The uterine epithelial cells then stop multiplying, mature, and become receptive to the embryo. This is a key step in the process of implantation.

<pubmed>PMC3320855</pubmed>

Lab 3

Question 1

The post-fertilization age (or conceptional age) is the time that has passed since fertilization of the egg. The gestational age, however, is measured from the first day of the woman's last menstrual cycle to the current date. A normal pregnancy can range from 38 to 42 weeks.

The gestational age is approximately two weeks greater than post-fertilization age. Gestational age is more clinically significant because its start date can be clearly determined both before and after birth, whereas the exact moment of fertilization must be inferred.

Post-fertilization and gestational age

Question 2

The somites developed from paraxial mesoderm, and will give rise to sclerotome, dermatome and myotome tissues.

The sclerotome relates to the axial skeleton and the proper functioning of the vertebral column: Sonic hedgehog signalling causes the ventromedial portion of the somite to differentiate into sclerotome.The sclerotome then develops into cartilage (chondrocytes) due to the transcription factor Pax 1.

Dorsolaterally, the dermomyotome develops first, which then differentiates into the dorsal dermatome and the ventral myotome.

The dermatome will contribute to the formation of the dermis due to the neurotrophin 3 factor. The dermis consists of: firstly, a more superficial papillary layer which has fine collagen and elastic fibres and contains small blood vessels (arterioles and capillaries), lymph and nerves. Secondly, a deeper reticular layer with dense collagen fibres and thick elastic fibres and it contains lymph, vascular plexus, nerves and appendages.

The ventral myotome can be split up into the epaxial myotome (dorsomedial quarter) and the hypaxial myotome (dorsolateral quarter). The epaxial myotome will result in formation of the erector spinae muscles and the hypaxial myotome will give rise to muscles of the trunk (ventrally) and limbs. The type of muscle which is formed is skeletal muscle - striated, multinucleated myofibers. Proteins such as Wnt 1 and 3 are related to the expression of genes which will cause muscle development.

Online Embryology course developed by the universities of Fribourg, Lausanne and Bern with the support of the Swiss Virtual Campus - Somite development.

Lab 4

Question 1

One of the invasive prenatal diagnostic techniques is chorionic villus sampling (CVS). A small sample of the developing placenta is obtained to test for genetic abnormalities. To collect this sample, a slender needle is inserted through the abdomen and into the placental tissue. The chorionic villi are then examined in a laboratory. CVS

Genetic abnormalities, such as Down Syndrome or Cystic Fibrosis can be tested for by this CVS diagnostic technique. It is generally performed between 10 and 12 weeks of pregnancy and has emerged as the only safe invasive prenatal diagnostic procedure prior to the 14th week of gestation. <pubmed>16533654</pubmed>

The other invasive prenatal diagnostic technique is amniocentesis. A sample of approximately 16 mL of amniotic fluid is collected from the amniotic cavity. To collect this sample, a long needle is inserted through the abdomen and into the amniotic sac. The embryo and the placenta remain untouched during the procedure. Ultrasound is often used prior to or during the procedure to locate the amniotic sac from which the sample is taken. The test is generally performed at around 16 weeks of prenancy. Amniocentesis procedure.

The fluid can be examined for fetal lung maturity, genetic evaluation and sex determination, the presence of infection, spina bifida and other neural-tube defects,or chromosome analysis to test for conditions such as Down syndrome. To screen for neural-tube defects and Down syndrome, blood tests can be performed. Elevated levels of the alpha feto protein may indicate a developmental abnormality. Amniocentesis testing

Question 2

The following paper describes a therapeutic use of umbilical cord stem cells:

<pubmed>22500090</pubmed>

It has long been known that mesenchymal stem cells can contribute to the alleviation of neurologic deficits. In this paper, researchers investigated the possible mechanisms which could underly the beneficial effect of human umbilical cord-mesenchymal stem cells on spinal cord injury.

Rats used in this experiment underwent surgery to induce neuronal damage. The skin and muscles overlying the thoracic cord were separated and retracted, the T9 vertebral level was removed by laminectomy, and the underlying spinal cord segment was exposed by slitting the dural sheath. A rod was placed above T9 and dropped from a height of 25mm to induce an incomplete partial SCI. Rats were then randomly assigned to different groups. Human umbilical cord blood was obtained from normal full-term pregnant woman. The mesenchymal stem cells obtained from the human umbilical cord blood were transplanted into the boundary zone of the injured site of some rats. Rats without the transplantation were in the control group. Animals received a daily injection of bromodeoxyuridine during the 7 days after treatment. Various experiments were carried out on both the experimental and the control groups and data of both was compared.

One of the tests looked at the area of the cavity of the damaged spinal cord. Through imaging it was found that the cavity volume was smaller in the rats with transplanted mesenchymal stem cells compared to the control group. The recovery of hindlimb function was also assessed. The motor function scores of rats with injected mesenchymal stem cells were significantly higher at 7 weeks after SCI, in comparison to the control groups. Scores demonstrated that the neurological function dramatically improved in treated rats. Thus, human umbilical cord blood-mesenchymal stem cell transplantation led to a significant improvement of behavior as well as the reduction of cavity volume after spinal cord injury.

Cells labelled with bromodeoxyuridine were counted in the ependymal and parenchymal regions. Proliferation of the newly generated cells increased greatly in treated rats as compared with the control rats. This demonstrated that the mesenchymal stem cells derived from the human umbilical cord blood could enhance proliferation of endogenous cells within the spinal cord. It was observed that both endogenous cell proliferation and oligogenesis contributed to functional recovery in the treatment group. Rats were also examined for immunoreactivities. Results showed weak responses for the control groups, yet high responses for the treatment group. This suggests that the presence of mesenchymal stem cells creates an influential microenvironment within the spinal cord. Furthermore, transplantation of mesenchymal stem cells protected injured spinal cord cells from apoptosis.

Taken all his data together, treatment of spinal cord injuries with human umbilical cord blood-mesenchymal stem cells has a neuroregenerative and a neuroprotective effect which could be therapeutically used to treat spinal cord injuries.