Ahmed Mahmoud

Past resaerch

On this page, you can find a brief overview of some of my work. For full PDF documents of the below research and more, click here.

Past research

Capstone Research 


The SRY Gene and Its Role in Sex Determination

Ahmed Mahmoud

Department Of Biology New Jersey City University, Jersey City NJ, 07305

                                                                        Submitted for review on 11/12/2009



The fate of a gonad is usually determined by the sex chromosomes in humans. The presence of a Y chromosome usually constitutes a male. A specific region on this chromosome is the specific male determining factor. The SRY gene, part of the SOX family of proteins works to inhibit specific functions of ovarian pathways to promote testis. Ovarian development is also guided by genes that work to activate the Wnt signaling pathway, which leads to a female. Rspo1 is one of those genes that have been proven to mediate the canonical b-Catenin pathway. The SRY gene has also been involved in Sex reversal syndrome due to translocation of the gene onto the X chromosome.

Keywords: SRY; SOX proteins; HMG; TCF; RSPO1; WNT4; b-catenin signaling pathway; Lef1 gene; Sex reversal; Gene translocation



The sex of an individual is controlled by the sex chromosomes in mammals (1). The gonads are ultimately the determining factor in humans (2). In humans, there are 46 chromosomes; two of them are the sex chromosomes that determine gonadal development (2). There are two types of sex chromosomes, the X and Y chromosomes. The X chromosome is the more gene packed of the two and is considerably larger in size then the Y chromosome (10). An XX set of chromosomes usually has gonads develop into ovaries (2). When there is a Y chromosome, the gonads develop as testes and a male is distinguished. Though the chromosomes the main carrier of



Genetic information, the Y chromosome has been fund to play a critical role in mammalian sex development (1).

What researchers have found is that on the short arm of the Y chromosome lies a gene called the SRY gene (Sex determining region of the Y chromosome)(3). It turned out that it was not the Y chromosome as a whole that determined the fate of the gonad but it was the SRY gene found on the Y chromosome that did.  In 1990, scientists found that the critical factor that leads to testis development was indeed the SRY gene (8). The default pathway of gonadal development leads to ovarian growth and the SRY gene is the genetic factor that pushes away from ovarian configuration to testicular development. The SRY gene does not work alone however. It is part of a family of proteins that have a domain that binds to DNA. This family is called the SOX family of proteins (8).  SRY is unique when compared to the other 19 SOX genes present in humans in that it does not have a transcription domain. No one knows yet what SRY’s target genes are when it binds to DNA but what is known is that SRY works closely with other SOX proteins particularly SOX 9. It has been seen that when SRY is no longer expressed for some reason, SOX9 takes over. Sox 9 is located on Chromosome 17 and spans 5401 pairs in length (Figure 1 A).


Female pathways

Just as SRY and the SOX proteins promote testis, there are a few players that determine an ovarian fate. R-Spondin 1(RSPO1) has become a key in the ovarian pathway. RSPO-1 becomes expressed more in females at the time of ovarian differentiation (7). RSPO-1 is a gene that codes for a secreted protein that activates a network of proteins used to produce WNT signaling molecules. This network is called the canonical b-catenin signaling pathway (3). Another key player is WNT-4 (wingless-related MMTV integration site 4). WNT-4 is a gene that codes for a protein that is involved in ovarian development (2). Both WNT-4 an RSPO1 are similar in length and are on the same chromosome, chromosome 1 (Figure 1 B & C).



Pathways of determination

With no presence of SRY, RSPO1 and WNT-4 work in tandem to activate the WNT-signaling pathway, which is required for ovarian development when in the presence of SRY, the WNT signaling pathway is blocked.  SRY Works close together with SOX9 to block the WNT pathway and promote testis formation (Figure 2). An experiment showing the repression of the WNT pathway was performed by Bernard Pascal and his team and is discussed further in the results section.

Sex reversal Syndrome

Sex reversal syndrome is a disorder, which creates a conflict between the gender of the chromosome and the gender of the gonads. This is when a male carries XX chromosomes or a female carries XY chromosomes. The incidence rate of SRS in humans is reported to be 1: 20000-100000 (5). SRS has been in the news for recently for its role in the Olympic games of the 1990’s. The first case of SRS came in 1936 at the Berlin Olympics. Two women by the names of Stella Walsh and Helen Stephens were sprinters that competed in the games that year. Rumors circulated that both these woman were actually men that competed as woman to gain an unfair advantage. Physically the woman appeared very masculine and had facial patterns that resembled a male (Figure 3).

After Stephens won the gold medal, Walsh accused Stephens of being a man. The Olympic committee at the time performed a very crude sex check involving a visual check of the external genitalia. It was confirmed after the check that Stephens was indeed a woman. A few years later however, Walsh was murdered and a post mortem check confirmed that Walsh had abnormal genitalia and different sex chromosomes.  After that incident, many other woman were accused of SRS.  In the 1960’s, the committee was forced to introduce a gynecologic examination. In 1968, Barr body detection was used after the committee found out that visual examinations were neither ethical nor useful. In the 1990’s, Polymerase Chain Reaction (PCR) was introduced and replaced the Barr body test. This PCR amplification was used for analysis of the SRY locus. PCR analysis for SRY was performed on all female competitors from 1992 on to 1999. In the 1992 Barcelona games, 15 out of 2000 tests were reported positive. In 1996, 8 out of 3000 were confirmed positive for SRY. The woman who tested positive were still allowed to play in Atlanta but it is not known if they were allowed to compete in Barcelona (6). 



One of the factors driving SRS is shown in a case study performed of a 20-year-old male who has undescended testicles. Upon initial examinations the phenotype of this individual resembled a normal male. There were a few slight differences in skeleton size, a lighter beard, and abnormal pubic hair differentiation.  The patient defined him self as a heterosexual and claimed his canonical erections and ejaculations were normal. A few tests were conducted and it was found that the patient had no uterus. Computer aided sperm analysis showed that no sperm was in his semen. After karyotype analysis, the patient showed 46, XX. He was born from a 37-year-old woman. During the pregnancy, she used old Chinese remedies to treat threatened abortion. The patient has two brothers and one sister that developed normally (5). It is unknown if they as well were subject to the same remedies that the patient was subject to in the womb. The experiment following these evaluations brings the event of gene translocation into play. Two methods were used in this analysis. The first was PCR amplification. DNA was first extracted from the patient and put in to PCR amplifications with a normal male and a normal human female. The second method was Fluorescent in situ hybridization of the DNA which just stains the DNA.



SRY blocks the WNT signaling pathway

The experiment used to look into the effect SRY has on the WNT signaling pathway used the TOPFLASH assay in three different kinds of cells. HEK293T (Human kidney cells), NT2/D1 (Human Carcinoma cells) and Hela cells. The TOPFLASH assay contains a binding site for TCF (T-cell specific HMG box factor) which is a protein found in the above-mentioned cells that play a role in WNT signaling. The control for TOPFLASH is FOPFLASH, which contains a mutation in which it cannot bind to TCF protein. BIO (6-broindirubin-3’-oxime is used to activate TOPFLASH. When BIO was added, it activated TOPFLASH in HEK293T and NT2/D1 cells but not in Hela cells which suggested that Hela cells do not play a role in the WNT pathway. Activation of TOPFLASH was reduced two times by SRY in HEK293T cells (Figure 4 A). This showed that SRY came in and blocked the Signaling pathway. In NT2/D1 cells, SRY also reduced activation but not as much as in the kidney cells.  To investigate SRY action, a plasmid that codes for SRY was added with a plasmid encoding for a mutant form of b-catenin called S37A. The S37A mutation has a more active form of b-catenin than its wild type. In HEK293T and NT2/D1 cells, the S37A mutation TOPFLASH very strongly when there was no SRY present. In the presence of SRY, there was no activation by S37A in HEK293T cells (Figure 4 B). This inhibition showed that SRY blocked the WNT signaling upstream of TCF.



RSPO-1 Activates b-catenin Signaling

Rspo1 is a major player in the Signaling pathway. This Signaling pathway is activated only in female development. To prove that Rspo1controls this pathway, an experiment was conducted Anne-Amandine Chassot and her team in which the levels of the Lef1 gene which is a gene found involved in b-catenin signaling.  Three different Urogenital ridges were stained for the Lef1 gene. The first was an XY positive for Rspo1. The second was an XX also positive for Rspo1 and the third was an XX negative for Rspo1.  High levels of Lef1 were found in XX gonads positive for Rspo1. In XX Rspo1 -/-, they observed no staining at all (3). The data suggested that When Rspo1 is present in the XX urogenital ridge, b-catenin signaling occurs and produces genes like Lef1 (Figure 5, Stain 3). When Rspo1 is not present in the XX urogenital ridge, b-catenin signaling does not occur and does not produce the Lef1 gene (Figure 5, Stain 2). When an XY gonad contains Rspo1, it still does not produce the Lef1 gene which indicates that b-catenin signaling is disabled (Figure 5, Stain 1). This could be due to the presence of the SRY gene and its inhibition of the signaling pathway.

SRS and Translocation

As shown above, the 20-year old male whose karyotype was 46 XX was subject to further analysis to determine why he had Two X chromosomes and still had all male characteristics except undesceneded testicles.  The DNA of the patient and control samples by PCR amplification showed that the patient and normal male amplification were the same. The SRY gene could be seen in both the patient and the normal male and the normal female contained no visual sign of SRY (Figure 6A).  Through Fluorescent in Situ hybridization, the short arm of the patients X chromosome showed a green hue that showed that the SRY gene was translocated to that particular Chromosome (Figure 6B).



The SOX proteins effect the b-catenin signaling pathway that is required for a variety of different functions but mainly for ovarian development. The main inhibitor of this pathway is SRY which is the main SOX protein. The experiment performed by Bernard Pascal and his team showed that SRY can block b-catenin signaling in human kidney cells. Many Sox proteins are known to block the signaling pathway but the way in which they inhibit it is not known. This might be due to the fact that there is confusion between the transcriptional functions of the SOX proteins. The inhibition of SRY was not as powerful in NT2/D1 cells or Hela cells. These cells express a number of male genes but may not be as useful in looking into the Wnt pathway (1).

In the experiment showing that Rspo1 is the mediator of the b-catenin signaling pathway, the urogenital ridges containing Rspo1 show staining for the Lef1 gene. When the signaling pathway is activated, b-catenin can interact with transcription factors such as Lef/TCF which can start up-regulation of specific genes like Lef1 (3). A very interesting part of this experiment was how the XY gonad positive for Rspo1 showed no staining for Lef1. This could be due to the fact that because SRY is presumed present, it is inhibiting the signaling pathway and preventing the regulation of the Lef1 gene. Translocation of the SRY gene results in an X chromosome with an SRY gene on it (9).  There are two phases in human sexual development; the first involves gonad formation, which could be regulated by the SRY gene, and the second is when the somatic cells change into genitals based on secondary sex characteristics (5). There are two types of XX male patients, SRY positive and SRY negative. As shown in the case, there are no abnormalities in the patients phenotype and can only be distinguished when they are infertile therefore it is very hard to find some one that is SRY positive before puberty so that further studied can be performed. In the case of SRY negative individuals, they can be diagnosed at birth due to abnormal genitalia.

There are many genes that play a role in gonadal development in both males and females. There is still much research left to be done about the sox family of proteins and their transcription functionality. In addition to SRY there are many others that play a role in testis development such as SOX3, SOX9, SOX10 and FGF9 (fibroblast growth factor 9)(5). The SRY gene is a founder of these proteins and plays a critical role in the inhibition of the ovarian pathway and as a start point for testis formation in which other SOX proteins such as SOX9 help to repress the ovarian pathway.











Figure 1A. Chromosome map showing location SOX9 gene on chromosome 17.



Figure 1B. Chromosome map showing location of Rspo1 on chromosome 1.


Figure 1C. Chromosome map showing location of WNT4 on Chromosome 1.



Figure 2. Diagram showing the different pathways to ovarian and testis development. Rspo1 and WNT4 promote b-catenin signaling that leads to ovarian development. SRY blocks b-catenin signaling and works closely with SOX9 to promote testis development.


Figure 3. Picture of Stella Walsh (left) and Helen Stephens (right). Were accused of being male to gain an advantage on the Olympics. After her murder, it was determined that Helen Stephens had an abnormality in Sex chromosomes and abnormal genitalia.





Figure 4 A. Shows TOPFLASH being reduced two fold when SRY is present. B shows S27A mutation and the affect of SRY repressing the b-catenin mediated activation on HEK923T cells.



Figure 5. Staining of three urogenital ridges. Stain 1 shows XY gonad positive for Rspo1with no stain. Stain 2 shows XX gonad also positive for Rspo1 with staining for Lef1 gene. Stain 3 shows XX gonad negative for Rspo1 showing no staining.



Figure 6 A. PCR analysis for SRY of SRS patient (marker 4) compared to Normal Male (marker 2), and normal Female (marker 3). Patient and normal male show the same amplification of SRY.



Figure 6 B. FISH (Fluorescent in Situ Hybridisation) of Patients chromosomes. Green fluorescence shows that SRY gene was translocated to the X chromosome.



1. Bernard, Pascal. (2008). Human SRY Inhibits β-Catenin-mediated transcription. Int J Biochem Cell Biol, 40(12), 2889-2900.

2.Nef, S., Vassalli, J. (2009). Complementary Pathways in mammalian female sex determination. Journal of Biology,8(74).

3.Chassot, A., Ranc, Fariba., Gregoire, E., Roepers-Gajadien, H., Teketo, M., Camerino, G., Rooij, D., Schedl, A. And Chaboissier, M. (2008). Activation of β-catenin signaling by Rspo1 controls differentiation of the mammalian ovary. Human Molecular Genetics, 17(9), 1264-1277.

4.Smith, C., Shoemaker, C., Roeszler, K., Queen, J., Crews, D. and Sinclair, A. (2008). Cloning and expression of R-Spondin1 in different vertebrates suggests a conserved role in ovarian development. BMC Developmental Biology,8(72).

5.Wang, T., Liu, J., Yang, J., Chen, J. And Ye, Z. (2008). 46, XX male sex reversal: a case report and review of the genetic basis. First Int. Journal of Andrology, 41, 59-62.

6. Ritchie, R., Reynard, J. And Lewis, T. (2008). Intersex and the Olympic games. J R Soc Med, 101, 395-399.

7. Marchal, J., Acosta, M., Bullejos, M., Diaz de la Guardia, R. And Sanchez, A. (2007). Genomics, 91,142-151.

8.Sinclair, A. (2001). Eleven years of sexual discovery. Genome Biology, 2(7), 4017.1-4017.3.

9. Newton, G. (2003). SRY and sex reversal. Retrieved October 1, 2009, from http://genome.wellcome.ac.uk/doc_WTD020752.

10. Brooker, R. J. (2009). Genetics Analysis & Principles. Minneapolis: McGraw-Hill.


Lentiviral Gene Therapy


Overview of X-Linked Adrenoleukodystrophy

The x-linked adrenoleukodystrophy or ALD is a brain disease that occurs in young boys. The disease is x-linked and caused by defects in genes (Cartier, et al., 2009). It occurs in mostly males because of the fact that males have only one X chromosome. If the male inherits an X chromosome carrying the gene defect, the male also inherits the disease. Females must acquire two defective X chromosomes and is why females rarely inherit this disease.  The disease is due to abnormalities of the ABCD1 gene that codes for the ALD protein. This protein functions as a peroxisomal remover of Very Long Chain Fatty Acids or VLCFAs. The VLCFAs accumulate in the brain where it damages the myelin sheaths of neurons in the brain. The boys who inherit this disease start to experience neuron damage as early as 6 years old and usually do not survive shortly after (Cartier, et al., 2009).

Previous studies and treatments

The study is based on a variety of work including previous work conducted on mice. In ALD deficient mice, results showed that they did not show any sign of symptoms for up to 6 months. Later in their life at about 15 months, mice began to show abnormal neurological symptoms (Pujol, et al., 2002). The only treatment proven effective against X-linked ALD is stem cell transplantation. This treatment however has its drawbacks. Hematopoietic Cell Transplantation or HCT is limited because of its strict requirements that involve very matching donors. The added risk of mortality due to the fact that HCT is only effective on young children (typically ages 2-3) makes it even more limited in its use. The new treatment being tested is called Hematopoietic Stem Cell (HSC) gene therapy. HSC uses lentiviral vectors to infect cells that do not normally divide such as CD34+ stem cells (Cartier, et al., 2009). Lentiviral vectors have shown to be a safer alternative to retroviral vectors because of the LV vector’s   lesser tendency to integrate into places where cancer may occur. Lentiviral Vectors have a self-inactivating design that essentially inactivates enhancers and promoters in its long terminal sections (LTRs) of the DNA, which support the safety of using these vectors (Cattaglio, et al., 2007).  Several viruses are used in lentiviral gene therapy. Some viral vectors such as simian immune deficiency virus (SIV), have been used in gene therapy targeting human stem cells. In that particular study the SIV lentiviral vector was used to temporarily correct human x-linked chronic granulomatous disease (X-CGD) that affects the function of phagocyte enzymatic activity.  X-CGD patient’s blood stem cells were infected with the SIV vector that contained the corrected gene. Six weeks later, the blood cells expressed the transgene (Nauman, et al., 2007).

Transfer of Corrected ABCD1 gene to HSCs

The vector used in this study is a replication defective Human Immune deficiency type-1 (HIV-1). The data for this study is based on two ALD patients who suffer from X-linked ALD. These two children ages 7.5 (P1) and 7 (P2), had no matching donors for HCT. Blood cells with nuclei were drawn out of both patients and then subjected to positive selection by immunomagnetic procedures. The HSCs isolated were then infected with the HIV-1 vector containing wild type ABCD1 that the two patients had mutations in. These HSCs were then frozen to allow for analysis and test on their replicative integrity. The transduced HSCs were then put back into the patients after full myeleoblative conditioning. 5 days after integration, 50% of P1 and 33% of P2’s HSCs respectively, showed expression of the ALD protein. The VLCFA levels were reduced in the HSC’s by 55% and 68% for P1 and P2 respectively (Cartier, et al., 2009).

The expression of ALD protein in corrected transplanted HSCs.

One month after the transplantation, ALD protein rates in peripheral blood cells showed an increase of 23% for P1 and 25% for P2. These percentages, however fell almost half around the 9 month stage. The ALD levels began to stabilize in both patients, leveling off at 10% at 24 months and 17% at 30 months for P1 and P2 respectively. VLCFA levels in peripheral blood cells showed a decrease by 20% for P1 and 28% for P2 20 months after transplantation (Cartier, et al., 2009).

Analysis of how the transplanted HSCs were distributed was done by analyzing the insertion sites of the Lentivirus through a type of Polymerase chain reaction known as linear amplification-mediated polymerase chain reaction (LAM-PCR). This system detects DNA sequences down to single cells. This aids in measuring the renewal of HSCs in and detects side effects to the gene transplantations (13). When performing these tests on CD14, CD15, CD3, and CD34 HSCs, the LAM-PCR showed a high amount of insertion sites that meant an even distribution of the lentiviral HSCs.  In finding out whether the HSCs had been infected, a comparison of insertion sites was conducted on CD3 T-Cells and CD19 B-cells as wells as myeloid cells of both patients. Cells from these two lineages (lymphocytes and myeloids), were compared to see if any of the insertion sites occurred in both lineages. The results showed that 4.6% of P1’s insertion sites and 1.4% of P2’s insertion sites occurred in both lineages. Compared to models, these results showed that the similarities in both lineages were much higher than the expected values, which indicates that the Lentiviral infection of HSCs had been accomplished (Cartier, et al., 2009).


Checking early signs of progress

            To monitor early progress of the therapy, researchers used LAM-PCR analysis to find out the amount of contribution of infected clones to gene correction (Schmidt, et. al., 2007). Identical insertion sites were counted and analyzed. The results showed that the distribution of clones was random and there were no dominant clones. Insertion sites that work on part of the same gene are markers indicating stem cell growth and proliferation. When these sites were analyzed results showed no significant differences (Cartier, et al., 2009).





The results of the patients after Gene therapy

Initial results

            Before the treatment, MRI scans of P1 revealed damage within sections of the brain. Lesions were found and allowed inflammation leading to disruptions of blood to the brain. These lesions stopped at 12 months after the therapy. The lesions in P2 were far more than in P1. The lesions in P2 continued to extend themselves further into the brain until 16 months after the therapy where at that point, became stabilized. Sensitivity in the aural pathways had disappeared as well. This indicated that the myelin damage had reversed. The results from both patients show a close comparison to patients treated by HCT but show a vast difference in untreated X-linked ALD patients who continually get worse (Cartier, et al., 2009).

VLCFA levels after treatment

In contrast to HCT patients that see a decrease of up to 55% in plasma VLCFA, levels decreased of the fatty acids in both HSC patients were reduced by 39% and 38% in P1 and P2 respectively.  These VLCFA levels reflect liver macrophages that have been subjected to the therapy since these macrophages and peripheral blood cells come from the same myeloid lineage that were transduced by the vector previously. The plasma VLCFAs decreased is considered a huge success despite the larger decrease in HCT patients. The results are considered a success because only 13 to 14% of blood cells expressed the corrected ALD gene. The success of the fatty chain reduction was also seen in transduced HSCs which suggests the there could have been ALD over-expression of blood cell precursors shortly after introduction of the vector (Cartier, et al., 2009). Researchers used real-time polymerase chain reaction (RT-PCR) test ABCD1 expression. RT-PCR is used to quantify specific DNA molecules, which is used to quantify the ABCD1 gene (VanGuilder, et al., 2008). The results of RT-PCR showed a 5 to 1 ratio of ABCD1 transgene to the mutated ABCD1 gene (Cartier, et al., 2009).

 Side effects after therapy

            Most side effects seen in both patients were in cognitive abilities. Left untreated, ALD patients suffer drastic continuous declines in cognitive functions.  P1 had been examined before therapy and showed a normal verbal intelligence reading but a nonverbal performance ability that was abnormal. After therapy, his Verbal performance shows no change but his nonverbal performance dropped drastically months after treatment and has slowly stabilized. P1 also had muscle weakness, which improved gradually until it completely subsided by month 14. P2 had fewer side effects showing no cognitive differences before and after treatment. The only side effect has become a visual defect that occurred 14 months after treatment and has stabilized (Cartier, et al., 2009).


Advantages over other Vector mediated treatments

            The study conducted here shows the superiority of using lentiviral vectors as opposed to other forms of vectors such as g-retroviral vectors. When using lentiviral HIV based vectors, the use of lab manipulation is minimized because of their ability to infect specific HSCs. Those HSCs remain at stable expression rates even when they do not have growth advantages. This is in contrast to the retroviral vectors that work on only immunodeficiency diseases where the transgene gains a growth advantage (Cartier, et al., 2009). Treatment with retroviral gene therapy also is a more invasive therapy that could lead to dangerous side effects due to its LTR activity that allows mutagenetic and carcinogenic effects. The self inactivating LTR of lentiviral vectors eliminates this cause for concern (Cattaglio, et al., 2007). Even self-inactivating retroviral vectors may not be as safe as lentiviral vectors because of the ability of lentiviral vectors to not contain an enhancer (Zhang, et al., 2007).

Lentiviral therapy leaves room for research

            In this study, the researchers used a variety of techniques to measure hematopoietic regeneration. Researchers did not find any evidence of mutation or early oncogenic effects but suggest that a longer study with a wider array of patients will be necessary to prove the genotoxicity in lentiviral HSC gene therapy is minimal. Past studies of ALD mice gave no insight to the amount of treated HSCs needed to see results. The only treatment used as a basis to assess HSC gene therapy was HCT which needed 80 % engraftment to yield a benefit. In this study, expression of corrected the ABCD1 gene yielded ALD protein in 15% of white blood cells, which come from the same lineage as bone marrow microglial cells (residing macrophages of the brain). These results are comparable to HCT presumably because of over-expression of the microglial cells that came from the infected HSCs (Cartier, et al., 2009).


            This study shows that allogenic HCT results can be achieved through HSC therapy. The use of a lentiviral vector to transduce cells seems to be a safer alternative to retroviral vectors which appear to affect hot spots such as oncogenes. Relative studies show that lentiviral viruses can be integrated in a massive portion of the stem cell genome. The benefits of this treatment come from the fact that patients do not need a donor with matching MHC molecules. HCT therapy can also be used on adults who suffer from X-linked ALD who develop symptoms. The advantage of this treatment over HCT is that HCT has a 40 % rate of mortality (Cartier, et al., 2009). Although not researched, HSC therapy could prove less lethal. This study lends support to the early work on somatic gene therapy and encourages further testing.








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