Case Teaching Notes for
“Cross-Dressing or Crossing-Over? Sex Testing of Women Athletes”

Introduction / Background

This case study is a “clicker case.” It combines the use of student personal response systems (clickers) with case teaching methods and formats. The case is presented in class using a series of PowerPoint slides punctuated by questions (called “clicker questions”) that students respond to before moving on to the next slide. In this way, students work their way through the material to understand (and solve) the problem presented in the case. Specifically designed for use in large introductory science classes, the method integrates lecture material, case storylines, student discussion, (clicker) questions, clarification of answers to those questions, more lecture, and data.

This case introduces students to the concepts of meiosis and crossing-over within the context of gender testing in female athletes. The “frame” for the case is the story of track athlete Santhi Soundararajan, whose sex questioned by officials at the 2006 Asian Games. Through Santhi’s story and the lecture material presented in class, students learn about the importance of meiosis in gamete formation, fertilization, and sex determination.

Developed for a one-semester, large lecture, introductory biology class for both majors and non-majors, the case would also be appropriate in an anatomy and physiology or endocrinology course. It requires that students have some existing knowledge about mitosis and nuclear division processes.

Objectives

• Explain the basis of sex determination in mammals.
• Describe the sources of variation in daughter cells produced by meiosis.
• Understand the role of the SRY gene in the determination of a human embryo as male.
• Explain how human sex reversal (XX/SRY+ male; XY/SRY- female) may take place.

Misconceptions

• A replicated chromosome with two chromatids is formed by fertilization, and has one maternal and one paternal chromatid.
• Meiosis involves two rounds of DNA replication.
• Sex can be deduced solely from a human karyotype.

Classroom Management / Blocks of Analysis

This case, as developed and outlined here, can be completed in a single 50-minute class period. Students work independently when answering clicker questions, although there is sufficient time to allow students to interact and discuss the case. The PowerPoint presentation includes animations of the processes of meiosis and crossing-over (see Supporting Materials under References, below). These animations each take about two minutes.

The case begins with the story of track athlete Santhi Soundararajan, then presents the idea of meiosis and meiotic separation of chromosomes, and then segues into the topic of crossover. After presenting crossover and SRY gender determination, the case shifts back to Santhi’s story, asking students what they would do if they were members of the committee at the Asian games assigned to determine whether Santhi is female. It ends with the current dilemma concerning what, if anything, athletic organizations should do about gender testing of athletes.

In-Class Case Presentation

In class, the instructor presents the case using a series of PowerPoint presentation slides punctuated by multiple choice questions that the students answer using their clickers.

Slide 1 is the title slide for the case.

Slide 2 introduces students to Indian track athlete Santhi Soundararajan. The slide includes a link to a video on the Internet that gives details about Santhi and the events which eventually led to her being stripped of the silver medal she won at the 2006 Asian Games after failing a sex test.

Slide 3 provides additional information about Santhi’s gender testing. At this point, instructors may want to present the historical context for gender testing in female athletes.

“Modern” sex testing in sports, by examination of the athlete’s chromosomes, was first introduced in 1966 by the International Amateur Athletic Federation (IAAF) in response to suspicion that several of the best women athletes from the USSR and Eastern Europe were actually men. These concerns became especially relevant during the Cold War when mistrust between the West and Eastern Bloc nations dominated the political arena. Sex verification testing was performed to ensure that males, who have naturally androgen-enhanced muscular strength, did not compete in women-only competitions. Prior to that, female Olympic competitors could be asked to parade naked in front of a board of examiners to confirm their sexual identity and to undergo gynecological exams.

Stanislawa Walasiewicz, a Polish athlete who won a gold medal in the women’s 100-meter race at the 1932 Summer Olympics in Los Angeles, was found to have male genitalia after her death in 1980. In a widely publicized case, a German athlete who had set a world record in 1938 in the women’s high jump was later found to be a male hermaphrodite. Sisters Tamara and Irina Press won five track and field Olympic gold medals for the Soviet Union and set 26 world records in the 1960s. Their careers suddenly ended when they failed to appear for sex testing at its introduction in 1966.

At the 1976 Montreal Olympics, all female athletes were subject to sex testing with the exception of Princess Anne, who competed for Britain’s equestrian team. Sex testing was done as recently as the 1996 Atlanta Olympic Games, but is no longer practiced in the Olympics. The International Olympic Committee voted to abandon these tests in 1999. However, the Olympic Council of Asia continues the practice.

Sex identity physical exams were soon augmented with sex-chromatin testing that looked for the one inactivated X chromosome, called a Barr body, in female cells. At the chromosomal level, a woman normally has two X chromosomes and a man has an X and a Y chromosome. Any abnormality in this number is a genetic disorder.

What about Santhi—is she a he? As students will learn later in the case, in December 2006 Santhi Soundarajan was stripped of the silver medal she won in the 800-meter race in the Asian Games. Reports of the exact results of Soundarajan’s sex test have not been released, but the tests she was given and their possible results are described on the Internet at: http://people.howstuffworks.com/gender-test.htm.

Slide 4 and Slide 5 ask students to discuss the criteria for determining whether an individual is male or female. The instructor should list these characteristics on the board. The most common distinguishing features are anatomical and physiological differences.

Slide 6 introduces the first clicker question of the case. Students are asked to imagine that they are members of the committee assigned to determine whether Santhi is female. The slide presents test results (made up for the purposes of the case; actual test results for Santhi Soundarajan are not known) and students are asked to indicate whether they believe she is female or male. Because athletes often fail to start or have irregular menstrual periods, the expected answer is female.

Slide 7—Instructors should explain that the next step in sex determination would be a chromosome test. You may want to mention that when a cell is not dividing, chromatin (unraveled chromosomes) is not distinctly visible.

Slide 8—In preparation for nuclear division, double stranded DNA condenses to form chromosomes to ensure proper separation of the genetic material. The DNA has already replicated during the S phase of the cell cycle so two chromatids are shown. The chromosomes are only visible in the nucleus when they are stained and condensed in preparation for nuclear division.

Slide 9—A karyotype is a picture of the condensed chromosomes in cells, arranged and numbered in order from largest to smallest size. Each chromosome also has a unique banding pattern when stained. A human has 23 pairs of chromosomes, with the X and Y chromosomes functioning as the sex chromosomes. Normally, an individual with an XX chromosome pair is female while an XY chromosome pair is male. The slide includes a link (http://learn.genetics.utah.edu/units/disorders/karyotype/karyotype.cfm) to a website that demonstrates how human karyotypes are made. It provides students with the ability to pair chromosomes from a smear and emphasizes the potential difference in the sex chromosomes.

Slide 10 presents a karyotype for “an athlete” and asks students whether the athlete is male or female. The correct answer (along with answers to other clicker questions in this case) is provided in the Answer Key.

Slide 11 requires students to think about the karyotype that Santhi would have if she were a normal female. Instructors should point out that sex testing is not so simple, and that it is important to understand the process of gamete formation, fertilization, and sex determination.

Slide 12, Slide 13, and Slide 14 introduce students to the concepts of gamete formation and fertilization.

Slide 15 demonstrates the difference between haploid and diploid chromosomes before and after DNA replication. A haploid cell where the DNA has not replicated has one chromatid for each chromosome but, if the DNA has replicated, it has two chromatids for each chromosome. A diploid individual has a maternal and a paternal chromosome of each type. The two members of a pair are called “homologous” chromosomes. If the DNA has not replicated, each chromosome has one chromatid. Following DNA replication, there are two chromatids for each chromosome.

Slide 16 checks students’ understanding of chromosome terminology, but connects the concept to the case.

Slide 17 and Slide 18 describe the process of meiosis. The DNA is duplicated prior to meiosis. Cells in the human body have 46 chromosomes, including 22 pairs of autosomes and one pair of sex chromosomes (XX in females, XY in males). Because there are two sets of chromosomes, one from each parent, the cells are diploid. Meiosis starts with a diploid cell and turns it into four haploid cells, cells with only one set of chromosomes. This means that when the chromosomes of egg and sperm cells combine at fertilization, the embryo regains the normal diploid number. This may be a good time to mention that the overall purpose of meiosis is to produce haploid cells and not all organisms undergo meiosis to produce gametes. Slide 18 includes a link (http://www.johnkyrk.com/meiosis.html) to an excellent animation demonstrating Meiosis I and II. Instructors may wish to revisit Slide 15 at this point and ask students to describe the chromosomes at each stage of meiosis.

Slide 19 and Slide 20 are clicker questions that are intended to review the process of meiosis.

Slide 21—At a genetic level, sexual reproduction is all about mixing up genes and putting together new combinations. Meiosis mixes up the parental genes in two major ways—independent assortment and crossing over. In independent assortment, each gamete gets only half the parental chromosomes. The exact combination in each egg or sperm can and does vary. This is because during meiosis the chromosomes assort independently, with a random member of each homologous pair going to each daughter cell. Slide 21 includes a link (http://wps.prenhall.com/wps/media/objects/…) to an animation that demonstrates independent assortment.

Slide 22 emphasizes the number of combinations from independent assortment without considering crossing over. With 23 pairs of chromosomes, there are 223 possible combinations for gamete formation with meiosis, or about 8 million possible combinations. One way to explain this is that, for each kind of chromosome, there are two choices, and since each choice is independent of each other choice, all the choices need to be multiplied together to get the answer.

Slide 23 and Slide 24—Sex determination in mammals depends on whether the sperm contains the X or Y chromosome. The egg only has the X chromosome.

Slide 25—The question on this slide leads students to the next concept, which is crossing over and swapping of the SRY gene.

Slide 26 and Slide 27—In crossing over, the members of each chromosome pair come together and swap segments. Crossover essentially makes completely new arrangements of the DNA in the two adjacent chromatids of homologous chromosomes.

Slide 28—The question posed in this slide checks students’ understanding of the crossing over material.

Slide 29, Slide 30, and Slide 31 describe the possible crossing over event in the male when the sex-determining region of the Y chromosome (SRY gene) ends up on the X chromosome. The video and animation linked to from Slide 31 (http://www.hhmi.org/biointeractive/gender/animations.html, see “Meiosis”) show the process of meiosis in a male. In the cell nucleus, chromosomes contributed by this male’s mother (in red) and father (in blue) pair up. For clarity, only the X and Y sex chromosomes and chromosome 5 of the 22 pairs of autosomes (nonsex chromosomes) are shown.

Each chromosome has replicated and consists of two identical chromatids. Crossing over can occur anywhere along the autosomes—and, here, they swap segments at each end. The X and Y chromosomes normally cross over only at their tips (indicated in blue on the Y). Note that SRY (the male Sex-determining Region on the Y chromosome) lies below this region.

The nuclear membrane breaks down, and the chromosomes line up along the cell’s equatorial plane and then move to the poles. A random member of each chromosome pair goes to each haploid daughter cell. A second division separates the chromatids and produces four cells, which develop into sperm.

Because males have one X and one Y chromosome, half the daughter cells formed by Meiosis I get an X and half get a Y. Following Meiosis II and sperm differentiation, half the sperm are X-bearing and half are Y-bearing. (In females, all the eggs will get one or the other X.)

In a general sense, the sex of the offspring is determined by the particular sex chromosome carried by the sperm. However, in the early weeks of development, all fetuses have preliminary structures for both sexes, and the immature gonads can become either testes or ovaries. In the seventh week of fetal development, the SRY gene on the Y chromosome activates and the gonads commit to becoming testes. In the absence of a Y chromosome and the signal to form testes, the fetus develops as a girl.

In the top panel of the animation, a sperm with an X chromosome fertilizes the egg; in the bottom panel, a sperm with a Y chromosome fertilizes the egg. The XX and XY fetuses develop along the same pathway through week six. Then SRY switches on; the XY fetus develops into a boy (bottom panel), and the XX fetus becomes a girl (top panel).

In rare cases, an XX individual develops as a male or an XY individual develops as a female. Researchers realized that studying the genes of these sex-reversed people would lead them to the master switch for sex determination. They subsequently identified a gene called SRY (Sex-determining Region on the Y chromosome) which is present only on the Y chromosome and controls male sex determination during embryonic development. In the unlikely event that the Y chromosome breaks below the SRY gene and transfers the SRY gene to the X chromosome during meiosis, the X chromosome then carries the gene that controls male development. This produces two sperm with abnormal sex chromosomes. When they fertilize eggs, the XX (SRY+) embryo develops into a boy (top panel), and the XY (SRY−) embryo develops into a girl. They are sex reversed.

Before the SRY gene was identified, scientists knew that there was a testes-determining factor on the Y chromosome that triggered the development of an embryo as a male. The challenge was to pinpoint its location. This was done by comparing, first, the observable physical structure of the chromosomes and, second, when the technology allowed, the DNA sequences of sex-reversed individuals with those of the normal population. (Sex reversal occurs in about 1 out of 20,000 births.) Screening with Y-specific DNA (DNA that is found only on the Y chromosome) showed that XY females tended to lack a certain segment of DNA on the short arm of the Y chromosome, whereas XX males carried DNA from that same region. Mapping that region yielded SRY. The protein encoded by SRY is apparently a transcription factor, and thus it regulates the function of another gene or genes.

The SRY gene, if it is present, sets in motion a cascade of biological activity that results in the development of a male. This genetic trigger directs the gonad—the primitive tissue from which both male and female sex organs derive—to turn into testes rather than ovaries. The testes then produce the hormones, including testosterone, that prompt the development of all other male-specific characteristics, including the external genitalia and, later, secondary characteristics such as facial hair and a deep voice. In the absence of a Y chromosome, the embryo will develop female structures.

The details of the genes that are involved in gonadal development are not shown in this case. However, if you are interested in explaining development in more detail, you should view the following link: http://web.nmsu.edu/~gene305/XXXY.jpg. There is another case that is very useful for explaining gonad development on the National Center for Case Study Teaching in Science’s web site called “Gender: In the Genes or in the Jeans? A Case Study on Sexual Differentiation” by William J. Hoese, California State University Fullerton, Judith Gibber, Columbia University, and Bonnie Wood, University of Maine Presque Isle (see: http://www.sciencecases.org/gender/gender_notes.asp).

Slide 32, Slide 33, and Slide 34 return the students to Santhi’s story. Clicker questions 10 and 11 are primarily student opinion questions, although the best answer to 10 is to perform additional tests. Although the SRY gene is apparently present in Santhi, there are many other genes that must be activated in order for normal male development to occur. If any of these other genes are mutated or nonfunctional, the embryo would develop as a female. It is probable that testing for only one gene is insufficient to address the question of sexual identity.

There are many other factors that ultimately determine the sex of an individual. For example, a condition called Complete Androgen Insensitivity Syndrome (CAIS) affects one in 20,000 individuals. In these cases, the mutated gene codes for the androgen receptor such that, although testosterone is present, it has no effect on cells. This mutation results in the development of female genitalia and characteristics. The scientific and medical authorities recognize CAIS individuals as females.

There have been reports that Santhi attempted suicide. The societal dimensions of Santhi’s story, a hero in her local community, is a topic beyond the scope of this case as it has been developed here, but it could be an interesting point for continued discussion. Other discussion topics that may be considered after completion of the case include drug testing in athletes, performance enhancement, and the genetics of homosexuality.

Assessment

This case was developed as part of an NSF-sponsored grant (# DUE 0618570) to determine whether clicker cases such as this one produced greater learning than the traditional lecture approach. As part of that project, the clicker cases had questions that were asked of students both before and after the class in which the material was presented. The questions were also used again during the final exam.

A transfer question was also developed for the case. This is a question designed to test whether a student could apply the knowledge that was given by the instructor in class to a new situation—a test of higher level thinking, according to Benjamin Bloom’s taxonomy of cognitive domain. This question, together with the additional pre- and post-case questions, are presented in the Answer Key.

Answer Key

Answers to the questions posed in the case study are provided in a separate answer key to the case. Those answers are password-protected. To access the answers for this case, go to the key. You will be prompted for a username and password. If you have not yet registered with us, you can see whether you are eligible for an account by reviewing our password policy and then apply online or write to answerkey@sciencecases.org.

References

Supporting Material

How Are Athletes Gender Tested?

http://www.ibnlive.com/videos/28851/how-are-athletes-gender-tested.html Last accessed: August 21, 2008

On the IBNLive News website from India, this short video reports on the events surrounding Indian athlete Santhi Soundararajan. Duration: 90 seconds.

Making a Karyotype

http://learn.genetics.utah.edu/units/disorders/karyotype/karyotype.cfm

A set of activities using Marcromedia Flash files to illustrate the creation of karyotypes from the Genetic Science Learning Center, University of Utah, http://learn.genetics.utah.edu.

Independent Assortment

http://wps.prenhall.com/wps/media/…

An Adobe Shockwave animation illustrating the principle of independent assortment. Duration: 2 minutes 10 seconds.

Meiosis

http://www.hhmi.org/biointeractive/gender/animations.html

This video with animation is narrated by David Page and shows a nucleus of a cell undergoing meiosis with crossing over. It clearly demonstrates the ability of the SRY gene to cross over to the X chromosome, leading potentially to the formation of XX (SRY+) males upon fertilization. Duration: 5 minutes 52 seconds.

Animated Outline of Meiosis

http://www.johnkyrk.com/meiosis.html

An animated outline of meiosis. Duration: 55 seconds.

Journal Articles

Allchin, Douglas. 2006. Male, female, and/or? How does nature define the sexes? The American Biology Teacher 68(6): 372–375.

Carlson, A. 1991. When is a woman not a woman? Women’s Sports and Fitness 13(2): 24–29.

Kindfield, A.C.H. 1991. Confusing chromosome number and structure: a common student error. Journal of Biological Education 25(3): 193–201.

Kindfield, A.C.H. 1994. Understanding a basic biological process: Expert and novice models of meiosis. Science Education 78(3): 255–283.

Ljungqvist, A., and J.L. Simpson. 1992. Medical examination for health of all athletes replacing the need for gender verification in international sports. Journal of the American Medical Association 267(6): 850–852.

Simpson, J.L., A. Ljungqvist, M.A. Ferguson-Smith, A. de la Chapelle, L.J. Elsas II, A.A. Ehrhardt, M. Genel, E.A. Ferris, and A. Carlson. 2000. Gender verification in the Olympics. Journal of the American Medical Association 284(12): 1568–69. Also online at: http://jama.ama-assn.org/issues/v284n12/rfull/jco00114.html

Books

Eugenides, Jeffrey. 2002. Middlesex. New York: Farrar, Straus, Giroux.

Fausto-Sterling, Anne. 2000. Sexing the Body: Gender Politics and the Construction of Sexuality. New York, NY: Basic Books.

Websites

David C. Page, M.D., HHMI investigator, member of the Whitehead Institute for Biomedical Research, and Professor of Biology at the Massachusetts Institute of Technology.

Home page: http://web.mit.edu/biology/www/facultyareas/facresearch/page.shtml

Biographical information about Dr. Page: http://www.hhmi.org/lectures/2001/page.htm

Summary of Dr. Page’s research from the HHMI website: http://www.whitehead.mit.edu/research/faculty/page.html

http://www.hhmi.org/research/investigators/page.html

How Cells Divide, NOVA Online, PBS

http://www.pbs.org/wgbh/nova/baby/divi_flash.html Last accessed: August 21, 2008

Side-by-side animations showing mitosis and meiosis.

Method for Identifying the Region of the Y-Chromosome Containing the Testis Determining Factor in Humans

http://zygote.swarthmore.edu/sex2.html Last accessed: August 21, 2008

Diagrams outlining methods used to identify and map SRY.

The Haploid-Diploid Cycle of Sexual Reproduction, Access Excellence

http://www.accessexcellence.org/RC/VL/GG/ecb/haploid-diploid_sexual_reproduction.php Last accessed: August 21, 2008

Overview flowchart of haploid/diploid, meiosis/mitosis in life cycle.

“Why would a female athlete fail a gender test?” Julia Layton

http://people.howstuffworks.com/gender-test.htm Last accessed: August 21, 2008

An excellent summary article, very similar to this case.

Slide Credits

Acknowledgements: This material is based upon work supported by the NSF Grant No. DUE-0618570. Any opinions, findings, conclusions, or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of NSF. The authors wish to thank Eric Ribbens, Peggy Brickman, Nancy Boury, Norris Armstrong, Nancy Rice, and Bill Rogers for their advice and contributions.

Date Posted: September 09, 2008.

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