Sex Determina)on of Medaka fish Victor Ando BIOL 354 Developmental Biology Department of Biological Sciences, Central Washington University, Ellensburg, WA Introduc)on • Scien)fic name Oryzias la)pes • Found naturally in Japan, Korea, China, Laos and Viet Nam • Size is around 30-­‐36mm • Prefer slow moving streams • Males are more slender and the dorsal and anal fins are larger than the females. Background Reproduc)on info • Spawning happens early in the morning • AVer fer)liza)on eggs will remain on the females genital pores un)l brushed onto aqua)c plants • Eggs usually hatch within 10 – 12 days • It is possible for medaka fish to reproduce within few days of producing the previous batch of eggs Significance of Medaka Dmy • Sex determina)on in medaka is male heterogame)c • Dmy is expressed only in the Y chromosome • Dmy is derived from Dmrt1 and share 93% similari)es • Gsdf gene is found in the tes)es of many fish species although it is thought that in Oryzias la)pes, Gsdf is downstream of Dmy and that the expression of Gsdf is dependent on expression of Dmy. Experimental design To determine the importance of Dmy for sex determina4on, they looked at 2 cases of wild medaka (Oryzias la)pes) with muta4ons in the Dmy gene. One case showed that when there was a frameshi? muta4on where a single nucleo4de inser4on in exon 3 of Dmy which caused all XY genotype to produce female phenotype in the mutant offspring. In the other case they found that when there was low levels of Dmy, large sum of the mutant popula4on came out female. •  In a recent study, researchers were able to determine that Dmrt1 and Figa is correlated to differen4a4on of gonads by looking at sex-­‐reversed medaka fishes. By looking at androgen-­‐
induced XX males, they were able to concluded that despite no Dmy was present (because there was no Y chromosome), the Dmrt1 was expressed similarly to that of a normal XY males. This finding would suggest that androgen is quite possibly what upregulates Dmrt1 expression in XX males therefore regulates tes4cular development. On the other hand estrogen-­‐induced XY females showed that although the expression of Dmy was present in the Y chromosome, ovarian produc4on s4ll proceeded because of the estrogen upregula4ng the Figa gene. From this experiment we can conclude that Dmy is a necessary component to sex determina4on, although its use in the process is to upregulate the Dmrt1 gene which subsequently regulates tes4cular development where as Figa regulates ovary produc4on. • 
Mechanisms of Sex Determina)on Dmrt1b(Y), simply known as Dmy is only expressed in male embryos. In species Oryzias la)pes, the presence of Dmrt1 is strongly related to testes development where as the Figa is related to ovarian development. Both the male and female medaka expressed similar levels of mRNA from Dmrt1 in the soma)c cells. During tes)cular development, Dmrt1 expression increases in the XY males and reaches same level as the Dmy. Expression of Dmrt1 and Figa are key component necessary for phenotypic differen)a)on of the gonads. Conclusion Looking at the results given by the sex-­‐reversed experiment we can see that gonad differen)a)on is purely based on the expression of Dmrt1 and Figa. The expression of Dmy in the XY genotype helps expression of Dmrt1 in male gonad produc)on by upregula)on. Future Direc)on In future experiments, it would be interes)ng to see the difference in regula)on of Japanese medaka which uses Dmy to its close rela)ve the Indian medaka that is regulated by Sox3. From this study we would be able to observe how evolu)on has allowed sex-­‐determina)on in different species of medaka to differ from one another over )me. Reference • Matsuda, M. (2003), Sex determina)on in fish: Lessons from the sex-­‐determining gene of the teleost medaka, Oryzias la)pes. Development, Growth & Differen)a)on, 45: 397–403. • Takehana, Y., Demiyah, D., Naruse, K., Hamaguchi, S., & Sakaizumi, M. (2007). Evolu)on of Different Y Chromosomes in Two Medaka Species, Oryzias dancena and O. la)pes. Gene)cs, 175(3), 1335–1340. hip://doi.org/10.1534/gene)cs.
106.068247 • Matsuda M, Nagahama Y, Shinomiya A, Sato T, Matsuda C, Kobayashi T, Morrey CE, Shibata N, Asakawa S, Shimizu N, Hori H, Hamaguchi S, Sakaizumi M (2002). DMY is a Y-­‐specific DM-­‐
domain gene required for male development in the medaka fish. Nature 417: 559-­‐563 • Takehana (2003), Co-­‐op)on of Sox3 as the male-­‐determining factor on the Y chromosome in the fish Oryzias dancena. Nature Communica)ons 5:4157 • Kondo M, Nanda I, Hornung U, (2003) Absence of the Candidate Male Sex-­‐Determining Gene dmrt1b(Y) of Medaka from Other Fish Species. Current Biology:416–420. Sex Determination of Parrotfish
Tykayah Baird
BIOL 354- Developmental Biology
Department of Biological Sciences, Central Washington University, Ellensburg, WA
Introduction
Common name – Parrolish Scien)fic name – Scaridae Lifespan – 5-­‐20 years, most not over 5 years Length – up to 4 feet Upwards of 80-­‐90 species Can change colors and paierns mul)ple )mes throughout life •  Live in warm tropical salt waters • 
• 
• 
• 
• 
• 
Figure 1: Female parrolish Experimental Design & Results
Sex change happens when a male is needed for: •  Reproduc)on •  A previous TP male is taken, dies , or leaves •  Protec)on EXP. 1-­‐ Expressed sequence tag (EST) sequencing and rapid amplifica)on of cDNA ends-­‐polymerase chain reac)on (RACE-­‐PCR) were used to iden)fy SOX3 and DMRT1 in groupers SOX3 – important in the sex determining region (related to that of the SRY on the Y chromosome) of the X chromosome in Parrolish DMRT1 (doublesex and male abnormal 3 (dsx and mab-­‐3)-­‐related transcrip)on factor 1)-­‐ important in tes)s development EXP. 2-­‐ By examining the gonads of upwards of 80 protogynous wrasse, the researchers were able to come up with a possible mechanism for development of sex determina)on (Figure 3). •  Parrolish are mostly all females un)l a male is needed •  Parrolish are protogynous hermaphrodites meaning they all start as females •  SOX3 is needed for matura)on of an ovary •  DMRT1 and testosterone are needed for the forma)on and transi)on to a tes)s Future Directions
Figure 3: Possible anatomy and development of protogynous hermaphordites as described by Munday, P et al. Mechanism of Sex Determination
Undifferen)
ated gonad Background
Reproduc)on •  Parrolishes can reproduce/ spawn year round but most oVen during summer months •  Females release thousands of eggs, males fer)lize eggs, eggs aiach to coral un)l birth Sexes •  Most species born all female first (protogynous) •  3 dis)nct phases •  Sexually immature drab juvenile females •  Ini)al phase (IP)-­‐ sexually mature males and females (all look and act like females) •  Terminal phase (TP)-­‐ sexually mature males who dominate reproduc)on •  Parrolishes have one of the most complex and unusual reproduc)on systems known to fishes Ini)al phase female •  Looking at more species to compare similari)es and differences between the sex determina)on-­‐ are all 90 species specifically protogynous? •  Assaying parrolish species with EST and RACE-­‐PCR as similarly done with groupers References
Immatu
re ovary Figure 2: Male parrolish Conclusions
Ini)al phas
e male DMRT1 expressed Presence of testosterone Termin
al phase male Figure 4: Sex Determina)on in ParroCish. The presence of the genes SOX3 and DMRT1 along with changes in androgens such as testosterone (natural and/or ar)ficial) are determining factors in the change of sexes in Parrolish. IP females and males may become TP males at any)me if a dominant male is needed for reproduc)on or protec)on. Abdel-­‐Aziz, E., Bawazeer, F., El-­‐Sayed Ali, T., & Al-­‐Otaibi, M. (2012). Sexual paierns and protogynous sex reversal in the rusty parrolish, Scarus ferrugineus (Scaridae): histological and physiological studies. Fish Physiol Biochem, 38(4), 1211-­‐1224. Animals -­‐ mom.me,. (2015). The Life Span of a ParroRish. 21 November 2015. hip://animals.mom.me/life-­‐span-­‐parrolish-­‐7878.html de Girolamo, M., Scaggiante, M., & Rasoio, M. (1999). Social organiza)on and sexual paiern in the Mediterranean parrolish Sparisoma cretense (Teleostei: Scaridae). Marine Biology, 135(2), 353-­‐360. Jonna, R. (2003). Scaridae, Animal Diversity Web. 29 November 2015. hip://animaldiversity.org/accounts/Scaridae/ Munday, P., Wilson White, J., & Warner, R. (2006). A social basis for the development of primary males in a sex-­‐changing fish. Proceedings Of The Royal Society B: Biological Sciences, 273(1603), 2845-­‐2851. Streelman, J., Alfaro, M., Westneat, M., Bellwood, D., & Karl, S. (2002). EVOLUTIONARY HISTORY OF THE PARROTFISHES: BIOGEOGRAPHY, ECOMORPHOLOGY, AND COMPARATIVE DIVERSITY. Evolu4on, 56(5), 961-­‐971. Zhou, L., & Gui, J. (2008). Molecular mechanisms underlying sex change in hermaphrodi)c groupers. Fish Physiol Biochem, 36(2), 181-­‐193. hip://dx.doi.org/10.1007/s10695-­‐008-­‐9219-­‐0 Sex Determination of The Tammar Wallaby
Tanner M Barnes
BIOL 354- Developmental Biology
Department of Biological Sciences, Central Washington University, Ellensburg, WA
Tammar Wallaby
Experimental Design & Results
•  Macropus eugenii •  Also known as the dama wallaby or darma wallaby •  Maximum recorded weight 9.1 kg in males, 6.9 kg in females •  Average lifespan in cap)vity 9.8 years, 14 years in the wild •  Australia, New Zealand, islands off coast of Australia •  These wallabies live in areas of dense vegeta)on with low trees and bushes, in thickets and around the outskirts of forests. The researchers collected gonads from wallabies at before, during, and aVer differen)a)on. RNA-­‐seq was used to verify expression of genes thought to be involved in gonadal sex differen)a)on, and discover novel genes. Transcriptome analysis showed differen)al up-­‐
regula)on in AMH, DHH, DMRT1, and SOX9 during development of males. FOXL2 was upregulated in female gonadal differen)a)on. Researchers added oestrogen to undifferen)ated gonads during gesta)on to determine its role in development. They found SOX9 protein was prevented from entering the nucleus. SRY and AMH were also suppressed. Germ cells from these XY gonads were capable of entering mitosis. Background
Reproduce sexually Gesta)on period is 25-­‐28 days One offspring per birth The joey is born under developed Females reach reproduc)ve maturity at 9 months, males reach maturity at 2 years -­‐  Sex determina)on is unique in that it does not occur in utero -­‐  The undeveloped offspring allow for easy manipula)on and observa)on of sexual differen)a)on Mechanism of Sex Determination
-­‐ 
-­‐ 
-­‐ 
-­‐ 
-­‐ 
AMH Sry AMH W
nt
4 Rs
F po
o 1 x
l
2
So
x9 Fgf9 Tes)s W
ntRs
4 po
1 S
o
x
9
Bipoten)al gonad XY gonad treated with oestrogen Foxl2 So
x9 Fgf
9 Ovary Figure 1: Sex Determina)on in Tammar Wallaby. Gene)c sex is determined by the XY chromosome mechanism. The SRY gene on the Y chromosome ul)mately determines sex through upregula)on of SOX9 and Fgf9 to promote tes)s development. Complete sex reversal was seen in XY gonads treated with oestrogen during gesta)on. Sex reversal is caused by suppression of SRY, AMH, and nuclear transloca)on of SOX9 protein. ER1 β
c
a W
t n
t
Rs
4 po
1 Conclusions
-­‐  Tammar Wallaby sex determina)on closely resembles the marsupial model. Gene)c sex determina)on follows the XY chromosome mechanism seen in humans and other eutherian mammals. In order for complete sex reversal to be seen researchers had to culture partum cells or add molecules during gesta)on. Masculiniza)on of ovary or demasculiniza)on of tes)s was observed from hormonal manipula)on postpartum. -­‐  Preven)on of Sox9 protein from entering the nucleus appears to be causing the sex reversal of XY gonads and may serve as a role in maintaining female phenotype. Future Directions
-­‐  Could combina)ons of hormones be used to cause complete sex reversal postpartum? -­‐  Further research on novel genes discovered in recent transcriptome study -­‐  Further research of ovarian development to uncover mechanism for complete sex reversal of XX gonad References
Pask AJ, Calatayud NE, Shaw G, Wood WM, Renfree MB. Oestrogen blocks the nuclear entry of SOX9 in the developing gonad of a marsupial mammal. BMC Biology. 2010;8:113. doi:
10.1186/1741-­‐7007-­‐8-­‐113. Renfree MB, Papenfuss AT, Deakin JE, et al. Genome sequence of an Australian kangaroo, Macropus eugenii, provides insight into the evolu)on of mammalian reproduc)on and development. Genome Biol 2011, (12):R81. Renfree MB, Shaw G. Germ cells, gonads and sex reversal in marsupials. Int J Dev Biol 2001, (45):557-­‐567. Pask AJ, Whitworth DJ, Mao C, et al. Marsupial An)-­‐Mullerian hormone gene structure, regulatory elements, and expression. Biol of Reprod 2004, (70):160-­‐167. Kwok, Jenny Y., "Exploring Sexual Differen)a)on in the Tammar Wallaby Using Transcriptomics" (2015). Master's Theses. Paper 728. hip://digitalcommons.uconn.edu/gs_theses/728 O’Hara et al. BMC Developmental Biology 2011, 11:72 hip://
www.biomedcentral.com/1471-­‐213X/11/72 Sex Determination and Parthenogenesis in Whiptail Lizards (Aspidoscelis)
Doug Bennett
BIOL 354- Developmental Biology
Department of Biological Sciences, Central Washington University, Ellensburg, WA
Introduction
Experimental Data and Results
Conclusions
The en)re genus Aspidoscelis (previously Cnemidophorus) exhibit some level of parthenogenesis. (source) Addi)onally, many members of this genus are hybrids as well as some having 3N and 4N chromosomal structure. For this review 4 species will be observed: A. inornatus, A. gularis, A. uniparens, A. 4gris, and A. neomexicanus. The focus will be on A. neomexicanus (New Mexican whiptail or Rio Grande Whiptail). Aspidoscelis neomexicanus Habitat: Low lying brush and grasslands, usually near rivers and creeks. Range: Southwest United States. Texas, New Mexico, and Arizona. Reproduc)on: Parthenogenic clutches of 2-­‐4 eggs Maximum Life Expectancy: ~6 years The first experiment performed first was to confirm that A. neomexicanus was a hybrid of A. 4gris and A. inornatus. Lowe and Wright (1966) performed a karyotype analysis by harves)ng leucocytes from whiptail bone marrow. The sister chroma)ds were isolated from c-­‐metaphase cells, then aligned on slides to produce figure 2. A. neomexicanus is unique in that it shows complete parthenogenesis with a 2N karyotype. At the same )me Lowe and Wright were interested in the karyotype of a 3N individual which represents a more common state for whiptails (figure 3). Lowe and Wright postulated that A. neomexicanus is a transi)on state from bisexual parental genomes to 3N parthenogenic species. Research into the genes and hormones controlling sex determina)on in the developing Whiptail are limited. However, a similar mechanism to other vertebrates is likely due to similarity in XX/XY sex determina)on. All observa)ons of hybridiza)on indicate sex is preserved with the ability of 2N hybrids to mate with other species to form further hybrids. Evolu)onarily parthenogenic species have shown no decrease in fitness due to lack of gene)c diversity. (Parker 1984) This indicates a poten)al increase in gene)c muta)on or a wide capability for developmental plas)city in whiptails. Addi)onally the ability to mate with a variety of other species or no other individuals at all and produce viable offspring gives A. neomexicanus an evolu)onary advantage. (Walker et al. 1990) Future Research
•  What genes control sex determina)on in Whiptails? •  Are there any environmental factors that contribute to sex determina)on? As stated above liile is know about the sex determining mechanism in these rep)les. Immunofluorescence targe)ng mammalian homologs of sox9 and foxL2 in developing gonads may be the first step in determining a mechanism. Also gexng a measurement of testosterone or estrogen produc)on in different )ssues would confirm common signaling pathways. qRT-­‐PCR could quan)fy aromatase produc)on in the developing gonad. Overall there is much to learn about sex determina)on in these organisms. Fig 2: Karyotype of A. neomexicanus (2N) and parental species A. inornatus (2N) and A. 4gris (2N). Photo credit Lowe and Wright 1966 Fig.1: Aspidoscelis neomexicanus in it’s natural habitat. Photo credit: Steven Richard Miller Fig 3: Karyotype of A. uniparens (3N) and bisexual parental species A. inornatus (2N) and A. gularis (2N) Photo credit Lowe and Wright 1966 Background
Parthenogenesis is the ability to reproduce without fer)liza)on. Only females can reproduce through parthenogenesis as males lacking eggs cannot carry/produce young. A. neomexicanus is the hybrid of A. inornatus and A. )gris (see experiments and mechanism for details). During this ini)al hybridiza)on event, both males and female A. neomexicanus were produced in a 50/50 ra)o however only females were viable as sexual reproduc)on is not possible between A. neomexicanus individuals. (source) This indicates gene)c sex determina)on as the likely mechanism. Parthenogenesis can take place through a variety of mechanisms. The New Mexico whiptail u)lizes ameio)c parthenogenesis. This means that the eggs produced by A. neomexicanus retain full 2N eggs, effec)vely cloning the individual. The results of these karyotype experiments show that despite the bisexual parents not having equal chromosomes, A. neomexicanus is a product of hybridiza)on in these two species. This lead Lowe and Wright (1966) to propose the mechanism of a 2N hybrid intermediate with a backcross to one of the parental species to lead to the many 3N species that are observed. A second study looked to determine if temperature dependent sex determina)on played any role in Whiptail reproduc)on. Crews (1989) took bisexual eggs from A. inornatus and parthenogenic eggs from A. uniparens. They were incubated at 25C and 31C, which were determined to be the extremes of the viable range from preliminary experiments. Individuals were sexed by microscopic observa)on of the developing gonads, the eggs were terminated before hatching at A. uniparens produced all female as expected, and A. inornatus resulted in a 1:1 ra)o of male to female at each temperature which is what is expected from complete gene)c sex determina)on. References
Crews, D. “Absence of Temperature-­‐Dependent Sex Determina)on in Congeneric Sexual and Parthenogene)c Cnemidophorus Lizards.” The Journal of Experimental Zoology 252, no. 3 (December 1989): 318–20. doi:10.1002/jez.1402520315. Lowe, Charles H., and John W. Wright. “Evolu)on of Parthenogene)c Species of Cnemidophorus (Whiptail Lizards) in Western North America.” Journal of the Arizona Academy of Science 4, no. 2 (1966): 81–87. IUCN. “Aspidoscelis Neomexicana: Hammerson, G.A., Lavin, P., Vazquez Díaz, J., Quintero Díaz, G. & Gadsden, H.: The IUCN Red List of Threatened Species 2007: e.T64278A12752324,” March 1, 2007. hip://www.iucnredlist.org/details/64278/0. Lynch, Michael, and Wilfried Gabriel. “Phenotypic Evolu)on and Parthenogenesis.” The American Naturalist 122, no. 6 (1983): 745–64. Parker, E. Davis, and Robert K. Selander. “Low Clonal Diversity in the Parthenogene)c Lizard Cnemidophorus Neomexicanus (Sauria: Teiidae).” Herpetologica 40, no. 3 (1984): 245–52. Walker, James M., James E. Cordes, and Ramadan M. Abuhteba. “Hybridiza)on Between All-­‐
Female Cnemidophorus Neomexicanus and Gonochoris)c C. Sexlineatus (Sauria: Teiidae).” American Midland Naturalist 123, no. 2 (1990): 404–8. doi:10.2307/2426570. Sex Determination of the Red-Eared Slider
Turtle, Trachemys scripta
Introduction
of Organism
Irina Cotes & Juliana Cuevas
BIOL 354- Developmental Biology
Department of Biological Sciences, Central Washington University, Ellensburg, WA
Experimental Design & Results
Red-Eared Slider Turtle, Trachemys scripta
Ø  Geographical regions: United States to Brazil,
Nearctic-Neotropical Regions
Ø  Habitat: Calm waters of soft bottoms with dense
vegetation of ponds, lakes, marshes, creeks, and
streams.
Ø  Diet: Feed mainly on plants and small animals such
as; snails, worms , aquatic insects and plants.
Ø  Life Span: 20-50 years
Male (26 degrees C)
-8-10 inches in length
-Thicker, longer Claws/Tails
-Sexually mature 2-5 years,
carapace length 4 inches
Female (31 degreesC)
-10-13 inches in length
- Sexually mature 5-7
years, carapace length
6-7 inches
Ø  Reproduction: Sexual maturity based upon Carapace
length.
o  Breeding season: late spring to early summer
o  Courtship: occurs underwater.
•  Male swims around female, vibrates his claws
around her face and head.
•  Receptive female swims towards male, sinks to the
bottom for mating.
•  45 min courtship, 10 min mating.
o  Nesting/Eggs: eggs laid March- June.
•  Amniotes, nest on soft sand with sun exposure.
•  Dug with hind feet 10-12 cm deep within 200 m of
water.
•  2-30 oval, soft shell eggs are laid.
Ø  Sex Determination:
o  Occurs during embryogenesis, Gonadal Sex
determined by temperature of egg incubation
during mid- trimester, TSP (Matsumoto et al,
2011).
o  Lack sex chromosomes , temperature-dependant
o  Female-producing temperature(FPT,31 degreesC)
o  Male- producing temperature (MPT 26 degrees C)
o  At 29.2 degrees C a 50:50 sex ratio produced.
o  A slight change in temperature can skew ratios/
cause population to crash (Matsumoto et al, 2011).
Experiment 2:
•  Can β- catenin rescue female development in
FPT gonads when exposed to aromatase
inhibitors?
•  FPT gonads at stage 17 were cultured in media:
control, AI, Li Cl, AI+Li Cl for 6 days.
•  Minimal Sox 9 expression is seen in the
gonads treated with AI alone.
•  No SOX9 in control, Li Cl, Al+Li Cl.
Experiment 1:
•  Investigate how estrogen affects gonad
development.
•  Immunocytochemical analysis of WT1,
LHX9, GATA4 and SOX9 expression
throughout the bipotential period
•  Subsequent sexual differentiation of the
gonads, with or without the application of
the β-estradiol or an aromatase inhibitor.
•  Estrogen treatment caused premature
suppression of SOX9 expression and
dissolution of cord structures in the
medulla.
•  Aromatase inhibition maintained SOX9
and testis cords and resulted in ovotestis
development.
Figure 1 Immunocytochemistry of FPT Stage 17 gonads. Cultured in media for
6 days. Containing Li Cl (promotes β- Catenin/Wnt) , AI (Aromatase inhibitor),
Al+LiCl, & untreated media. Activation of Wnt signaling rescued female
development in the absence of Estrogen. Taken from Mork et al. 2013. Mechanism of Sex Determination
Bipotentia
l Gonad
Female
Temperature 31
degrees C
Male
Temperature 26
degrees C
Dmrt1
Arom
atase
FoxL2
SOX9
Testis
Differentiation
Conclusions
•  Temperature- dependent sex determination.
•  Dmrt1 and SOX9 required for testis formation.
•  FoxL2, Aromatase, Rspo1, Wnt-4, β- catenin
required for ovary formation.
Experiment 1:
•  Endogenous estrogen feminizes the medulla of
bipotential gonad by inhibition of SOX9
expression.
•  Inhibition of Estrogen receptor signaling delayed
the SOX9 down regulation in the differentiating
ovary at the end of the bipotential period.
•  Treatment with β- estradiol induced developed
down-regulation of SOX9 but did not affect
WT1,GATA4 or LHX9 expression.
Experiment 2:
•  Activation of Wnt-4 signaling rescued aspects of
female development in absence of estrogen.
•  Wnt-4 not likely to act upstream of estrogen in
gonad.
•  β- Catenin downregulation of SOX9 must occur
downstream or independently of estrogen.
Future Directions
Experiment 1:
•  How does sex reversal in wild populations exposed
to environmental estrogens affect the red eared
slider turtle?
•  Look at the relationship between SOX9 and
estrogen in these turtles.
Experiment 2:
•  Does estrogen promote SOX9 downregulation and
female development in MPF turtles?
•  Could be tested by using MPT gonads with
estrogen and a Wnt-4 inhibitor to see if SOX9
expression would be high in the absence of Wnt-4
signaling.
•  Not a lot of Wnt-4 inhibitors known in turtles.
References
βcatenin
Rspo1
Barske, L.A., Capel, B., 2010. Estrogen represses SOX9 during sex determination in the redeared slider turtle Trachemys scripta. 341, 305-314.
Mork, L., & Capel, B. (2013). Conserved action of β-catenin during female fate determination
in the red-eared slider turtle. Evolution & Development, 15(2), 96-106. doi:10.1111/ede.12020
WNT4
Ovary
Differentiation
Figure 1: Sex Determination in the Red- Eared Slider Turtle. At Male Specific Temperature, the female genes
are suppressed. Dmrt1 is activated. Dmrt1 then activates SOX9. The bipotential gonad will then differentiate into a
testis. At Female Specific Temperature, FoxL2 is activated and inhibits SOX9 expression. FoxL2 then activates
Aromatase. Rspo1 activates β- catenin and Wnt-4. β- catenin also inhibits SOX9 expression and there is ovary
differentiation in the bipotential gonad. This mechanism was modified from Yuiko et al., 2011. Matsumoto, Y., & Crews, D. (2011). Molecular mechanisms of temperature- dependent sex
determination in the context of ecological developmental biology. Molecular and Cellular
Endocrinology. doi:10.1016/j.mce.2011.10.012
Red-eared Slider. (n.d.). Retrieved December 1, 2015, from
http://naturemappingfoundation.org/natmap/facts/red-eared_slider_712.html
Red Ear Slider Turtle. (n.d.). Retrieved December 1, 2015, from
http://allturtles.com/red-ear-slider-turtle/
Sex Determination of Monodelphis Domestica
Matthew Everett and Elaina Martinez
BIOL 354- Developmental Biology
Department of Biological Sciences, Central Washington University, Ellensburg, WA
Introduction/Background
•  Scien)fic name-­‐Monodelphis domes4ca •  Common name-­‐ grey short-­‐tailed opossum •  Habitat -­‐ tropical, rainforest, grasslands, scrub and also human dwellings •  Lifespan: 3-­‐6 years (wild) 6-­‐10 years (cap)vity) •  Age of reproduc)ve maturity -­‐ 5 to 7 months •  Ma)ng behavior )ed to olfac)on •  Gesta)on -­‐ 14-­‐15 days •  1 to 15 offspring per liier, average of 4 liiers per year (Moore, 2006) •  Pups born in an embryonic stage of development makes them ideal canidates for lab experiments •  Most common animal used for research of marsupials (VandeBerg, 1997) Figure 1 Adult female M. domes4ca with offspring. (Weslall, 2012) Figure 2 Newborn M. domes4ca pups clinging to mother (Weslall, 2012) Mechanism of Sex Determination
3β-­‐HSD Future Directions
??? Development of scrotum ?? Estrogen Figure 3 Sex Determina)on in grey short-­‐tailed opossums. Sry gene on Y-­‐chromosome ac)vates the Sox9 gene. Sox9 then ac)vates expression of itself and other genes required for tes)s development such as AMH, and Fgf9, while inhibi)ng expression of genes needed for ovarian development such as β-­‐catenin. It is possible that Sox9 also inhibits the expression of Foxl2, which is another protein involved in ovarian development. Development of scrotum may be dependent on the androgen 3β-­‐HSD (Russell et al, 2003). Wnt4 and Rspo1 genes are ac)vated in the female; this ac)vates β-­‐catenin expression which inhibits Sox9. It is thought that Foxl2, ERα, ERβ, and possibly estrogen inhibt Sox9 expression as well; further studies are needed to dtermine this. Modified from (Sekido et al, 2009) Birth Experimental Design
& Results
Experiment 1: Estrogen (Moore, 1990) •  Conducted to find effect of estrogen on sexual development of opossum pups 0-­‐14 days aVer birth •  34 pups coated with 1-­‐2 μg oestradiol daily. Killed and karyotyped at various stages of development, some allowed to reach maturity •  Oestradiol inhibited tes)s and sex cord development in male pups •  Internal and external genitalia completely feminized except for scrotum •  Behavior of treated males at maturity was also feminized •  No effect on female pups Experiment 2: Androgens in scrotal development (Russell et al, 2003) •  Experiment on the role of androgens in scrotum development contradicts previous studies that men)on scrotum development is an androgen independent process. •  Immunocytochemistry was carried out to detect 3β-­‐
hydroxysteroid dehydrogenase (3β-­‐HSD), and androgen receptor (NCL-­‐ARp) throughout scrotal development •  Levels of 3β-­‐HSD in gonads and adrenals at embryonic day 14.5 are the same in both male and female •  Androgen receptor present in male scrotal )ssue, not present in females Conclusions
•  Sex of M. domes4ca is determined by sex-­‐
chromosomes (XX for female, XY for male) •  Sex-­‐determining region (Sry) on Y chromosome ini)ates male development •  Mechanism for estrogen inhibi)on of male gonadal development is unknown. •  Androgen 3β-­‐HSD plays a role in scrotal sac development, however the extent of role is unknown Scrotal bulge present in male. Wolffian duct present in both male and female. Day 1-­‐6 Müllerian duct forms in both male and female Day 10-­‐19 Day 14-­‐19 Müllerian duct regresses and gonad mesonephros degrades in male Wolffian duct regresses and gonad mesonephros degrades in female Day 24-­‐28 Day 19-­‐25 Tes)s descend into final posi)on at base of scrotum. Vas deferens differn)ates from Wolffian duct Uteri, oviducts, lateral vaginae, and vaginal culs-­‐de-­‐sac differen)ate from müllerian duct Figure 4: Timeline of postnatal development in M. domes4ca. Wolffian duct is present at birth but does not regress in females un)l two weeks postnatally. Müllerian ducts form postnatally and then regress in males star)ng day 10 aVer birth. Female and male reproduc)ve organ development is not complete un)l postnatal days 25 and 28, respec)vely. (Mackay et al, 2004) •  Oestradiol treatment along with immunocytochemistry staining with an an)body of a protein known to play a role in male sex determina)on (such as SOX9) in order to determine the mechanism behind the inhibi)on of tes)s development. •  Experiment using androgen receptor (NCL-­‐ARp) knockout mice in order to determine the extent of androgen-­‐dependence in scrotal development. References
Mackay, S., Xie, Q., Ullmann, S. L., Gilmore, D. P., & Payne, A. P. (2004). Postnatal development of the reproduc)ve system in the grey short-­‐
tailed opossum, Monodelphis domes4ca. Anatomy & Embryology, 208(2), 121-­‐133. doi:10.1007/s00429-­‐004-­‐0386-­‐1 Moore, D. (2006). "Monodelphis domes)ca" (On-­‐line), Animal Diversity Web. Accessed November 25, 2015 at hip://animaldiversity.org/
accounts/Monodelphis_domes)ca/ Moore, H.D.M., & Thurstan, S.M. (1990). Sexual differen)a)on in the grey short-­‐tailed opossum, Monodelphis domes4ca, and the effect of oestradiol benzoate on development in the male. Journal of Zoology, 639-­‐658 Russell, A. J., Gilmore, D. P., Mackay, S., Ullmann, S. L., Baker, P. J., & Payne, A. P. (2003). The role of androgens in development of the scrotum of the grey short-­‐tailed Brazilian opossum (Monodelphis domes)ca). Anatomy & Embryology, 206(5), 381-­‐389. doi:10.1007/
s00429-­‐002-­‐0300-­‐7 Sekido, R., & Lovell-­‐Badge, R. (2009). Sex Determina)on and SRY: Down to a Wink and a Nudge? Trends in Gene4cs, 25 (1), 19-­‐29. VandeBerg, J.L., & Robinson, E.S. (1997). The Laboratory Opossum (Monodelphis domes4ca) in Laboratory Research. Oxford Journals 38(1), 4-­‐12. Weslall, S. (2012). "Marsupials" (on-­‐line), Natural History Web. Accessed November 26 2015 at hip://retrieverman.net/category/
marsupials-­‐3/ SEX DETERMINATION OF KOMODO DRAGONS
Paola Rodriguez and Logan Gilbert
BIOL 354- Developmental Biology
Department of Biological Sciences, Central Washington University, Ellensburg, WA
Experimental Design & Results
Molecular Sex Determina)on of Cap)ve Komodo Dragons Introduction
Komodo dragons (Varanus komodoensis) are one of the world’s largest rep)les. Na)ve to the Indonesian islands of Komodo, Rinca, Flores, Gili Motang, and Pada, komodo dragons are well known due to their deadly salvia. Consis)ng of 50 strains of bacteria, their saliva can make just about any animal die from blood poisoning. •  Sex is hard to determine due to the difficulty in iden)fying their sex organs. •  Reach sexual maturity between the ages of 9 (females) and 10 (males) years old •  Mate between May and August, lay eggs in September •  Can live up to 30 years old in wild •  Have the ability to reproduce sexually and parthenogene)cally. •  They are listed as vulnerable by the IUCN. In this experiment, scien)sts analyzed DNA fragments in cap)ve komodo dragons in order to determine their sex for breeding programs. To do this, researchers took blood samples and ran them through a process of polymerase chain reac)on (PCR) amplifica)on using species-­‐specific nuclear DNA markers. In males, the PCR gels showed two alleles A and B overlapping into a single strand, indica)ng no difference between them. The males also showed a C strand which the females did not. Females had a longer, more intense A band, while the B band was not always visible. From this, researchers were able to differen)ate the homogame)c males from the heterogame)c females due to the difference in chromosomal bands. Parthenogenesis In 2007, a female komodo dragon in the London Zoo, UK, had produced a clutch of four viable eggs, despite not having been kept with a male in 2.5 years. Scien)sts wanted to know if this was caused by long-­‐term sperm storage or parthenogene)c reproduc)on, so they analyzed the DNA of the offspring and compared it to the mother via gene)c fingerprin)ng. The results showed that the mother and the offspring had iden)cal genotypes, so the possibility of a father being involved in this par)cular instance of reproduc)on was incredibly low (the chance of such an event happening in sexual reproduc)on is 0.0001). A follow-­‐up experiment comparing the alleles of the offspring to possible fathers confirmed the parthenogenesis hypothesis. Mechanism of Sex Determination
Background
•  It is noted that female komodo dragons do not have a ZZ chromosomes but instead have a WZ chromosome. Male komodo dragons have a ZZ chromosome pair. When reproducing pathogenically, the embryo will either be a WW or ZZ. However, WW embryos will die off while the ZZ embryo survives, making it so all of the offspring will be male. •  The way that scien)st determine the lizards sex is by ultrasound, blood work, laparoscopy of the gonads via endoscope, and radiography of the cloacal region. •  It has been recorded that a komodo dragon can reproduce without a male dragon. The offspring are not clones due to the fact that they hold only half the mothers DNA. Female No males → Parthenogenesis WZ = female → Asexual → WW or ZZ embryos → WW dying and ZZ survive Males → Sexual reproduc)on WZ and ZZ embryos Parthenogenic zygote → fusion of egg with polar body → polar body acts as sperm and restores diploidy = terminal fusion → offsprings are not clones of mother = male offsprings FIGURE 1: Comparison of normal and parthenogene)c fer)liza)on processes using sharks as a model hip://www.ansci.wisc.edu/jjp1/ansci_repro/misc/project_websites_08/tues/
Komodo%20Dragons/what.htm FIGURE 2. Another look at the process of parthenogenic fer)liza)on Conclusions
Unfortunately, as of right now very liile is known about the sex determina)on process of komodo dragons. What we do know is they use a ZW sex determina)on system, allowing them to reproduce both sexually and parthenogene)cally and making it possible for them to reproduce even in a small popula)on. Future Directions
While we know how komodo dragons reproduce, we don’t really know how their sex is determined during development. In order to get an idea what factors might be responsible for sex determina)on, we propose experiment comparing blood samples taken from male komodo dragons to blood samples taken from females. To do this, we would use a method like PCR amplifica)on to isolate enzymes abundant in each sex’s blood, and compare the different enzyme levels to each other. Once the differences in enzyme abundance have been iden)fied, future experiments would be able to research each enzyme’s role in a komodo dragon’s development. References
Sri Sulandari ; Moch Samsul Arifin Zein ; Evy Ayu Arida ; Amir Hamidy,01 June 2014, HAYATI Journal of Biosciences,Vol.21(2), pp.
65-­‐75 Molecular Sex Determina)on of Cap)ve Komodo Dragons (Varanus komodoensis) at Gembira Loka Zoo, Surabaya Zoo, and Ragunan Zoo, Indonesia Morris, Patrick J. ; Jackintell, Lori A. ; Alberts, Allison C. 1996, Zoo Biology, Vol.15(3), pp.
341-­‐348 Predic)ng the gender of subadult Komodo dragons (Varanus komodoensis) using two-­‐dimensional ultrasound imaging and plasma testosterone concentra)on Wais, Phillip C.; Buley, Kevin R.; Sanderson, Stephanie; Boardman, Wayne; Ciofi, Claudio; Gibson, Ricahrd. Nature. 12/21/2006, Vol. 444 Issue 7122, p1021-­‐1022 Parthenogenesis in Komodo Dragons 10 Chromosomes of Platypus Development
Alessandro Girardo
BIOL 354- Developmental Biology
Department of Biological Sciences, Central Washington University, Ellensburg, WA
Experimental Design & Results
Introduction
Ornithorhynchus ana)nus Duck-­‐billed platypus Average life span-­‐ 12 years Habitat-­‐ Lakes, Rivers, and Streams mountainous highlands to tropical rainforests •  Located on the eastern coast of Australia and island of Tasmania • 
• 
• 
• 
• 
Background
•  Breeding Season June-­‐September •  Males sexually mature at 2 and females at 3 years •  Male platypus use venom during ma)ng season •  Female platypus are egg laying mammals •  Reproduce by sexual reproduc)on •  Females nest eggs (1-­‐3 eggs)10 days under tail •  AVer 1 month developing in utero •  contain 10 sex chromosomes XX or XY pairs •  homology to birds, rep)les, and mammals •  evolu)onary between rep)les and other mammals in phylogeny •  shows an independent switch from zw to xy Iden4fying sex-­‐chromosomes in the platypus (Gruntzer 2004) •  Platypus chromosomes were prepared for viewing by flow sor)ng, chromosome pain)ng, and fluorescence staining •  to iden)fy platypus chromosomes and determine their homology rela)onships •  Chromosome specific paints iden)fied 21 paired chromosomes and 10 unpaired chromosomes named E-­‐1 through E-­‐10 •  Fluorescence dye indicated females contained unpaired chromosomes E1,E2,E5,E7, and E9 as 5 paired X chromosomes •  The male was iden)fied to have 5 pairs of xy chromosomes •  X1Y1X2Y2X3Y3X4Y4X5Y5 male mito)c chain is unique to monotremes Iden4fying sex determina4on genes in platypus (Gruntzner 2009) •  SRY is a mammal sex determina)on gene in but nonexistent in platypus •  RT-­‐PCR (reverse transcrip)on-­‐ polymerase chain reac)on) used to reverse to DNA process and view cDNA •  cDNA made from mRNA that binds to specific primers to form organs •  DNA heated then a specific primer was added that would bind to certain mRNA of that gene •  Taq polymerase aiached to the primer and added nucleo)des to elongate and extend the cDNA to view for analysis •  It was found that the DMRT1 and DMRT7 genes were expressed in the tes)s but not ovary •  WT1 genes expressed during process in the ovary but not tes)s Mechanism of Sex Determination
•  X1X1X2X2X3X3X4X4X5X5 Female platypus sex chromosomes •  X1Y1X2Y2X3Y3X4Y4X5Y5 Male platypus sex chromosomes . Figure 1. Picture on leV displaying chromosome pain)ng to iden)fy platypus( sex chromosomes (Gruntzner) Picture on right displays all platypus chromosomes found (Gruntzner) Mechanism of Sex Determination
(continued)
•  DMRT1 and DMRT7 genes shown to be expressed in high levels during tes)s development •  WT1 genes shown to be expressed in high levels for ovary development hip://imgur.com/gallery/zpb6Q1j Figure 2. Chart showing expression of )ssues in male and female platypus, showing what genes are involved in expressing reproduc)ve organs (Gruntzner) Conclusion/Future Directions
•  WT1 is a sex determina)on gene for ovary development in platypus, what genes that may regulate WT1 to determine female genes? •  5 pairs of sex chromosomes female 5 XX and male 5 XY •  DMRT1 and DMRT7 genes used to determine development of tes)s in platypus, what genes regulate the DMRT genes to determine male genes? •  Further experiments can be done where a knockout platypus could have WT1 removed if female or a DMRT gene removed if male to see what impacts there will be on reproduc)ve organs, to see if these genes are truly needed for reproduc)ve development? References
•  Grtutzner, Frank, Enkhjargal Tsend-­‐Ayush, and Diana Hamdan. "Characterisa)on of ATRX,DMRT1, DMRT7, and WT1 in the Platypus (Ornithorhynchus Ana)nus)." Reproduc4on, Fer4lity and Development 21 (2009): 985-­‐91. Print. •  Grtutzner, Frank, Fredric Veyrunes, and Paul Waters. "Bird-­‐like Sex Chromosomes of Platypus Imply Recent Origin of Mammak Sex Chromosomes." Genome Research 18 (2008): 965-­‐73. Print. •  Grtutzner, Frank, Patricia O' Brien, and William Rens. "Resolu)on and Evolu)on of the Duck Billed Platypus Karyotype with an X1Y1X2Y2X3Y3X4Y4X5Y5 Male Sex Chromosome Cons))u)on." PNAS 101.46 (2004): 16257-­‐6261. Print. •  Ocenanwideimages.com, Platypus swimming photo. •  Imgur.com/gallery/zpb6Q1j, Baby platypus photo. SEX DETERMINATION IN THE WESTERN HONEY BEE (APIS MELLIFERA) Apis mellifera drone. (Mid-­‐Atlan)c). Raegan Nelson BIOL 354: Developmental Biology Department of Biological Sciences, Central Washington University, Ellensburg, WA INTRODUCTION: LIFE IN A COLONY HAPLODIPLOID SEX DETERMINATION ♀ Live in social colonies with: •  Queen •  one per colony •  lives up to 5 years •  pheromones regulate colony •  can lay 1500 eggs/day •  Workers •  sexually immature females •  live up to 6 months •  build and protect hive •  forage for pollen/nectar Figure 1. An Apis Western mellifera dH
rone. All Bdee rones oney within a colony are male. The only purpose •  rear developing young (Apis mellifera) of a drone is to mate with a queen. (Mid-­‐
•  Drones Atlan)c). •  sexually mature males •  sole purpose: mate with queens •  begin ma)ng at 8 days old •  die aVer ma)ng or when expelled from hive in winter (Honey Bee and Schurko 2013) •  Queens mate in flight with mul)ple drones, away from home hive ♂ Csda/Csdb Csda/Csda or Csda ♀ fem ♂ fem Fem Am-­‐tra2 ♀ Am-­‐dsx ♀ development MATING RITUALS EXPERIMENTAL SUPPORT (CONT.) ♂ Am-­‐dsx ♂ development Figure 4. Mechanism for sex determina4on in the honeybee. Females develop from fer)lized eggs that are heterozygous for the complementary sex determiner (csd) gene. The csd gene codes for an ac)ve Csd protein that regulates female-­‐specific splicing of feminizer (fem) gene mRNA transcripts along with another gene, transformer2 (tra2). This splicing paiern codes for a func)onal Fem protein, which also partners with tra2 to regulate female-­‐specific splicing of Apis mellifera doublesex (Am-­‐dsx) gene. Fem has a autoregulatory loop for itself, synthesizing its own protein. Lastly, Am-­‐dsx codes for Dsx protein, leading to female development. Male honeybees are either homozygous or hemizygous for csd. This causes Csd ac)vity to be absent and results in an early stop exon during splicing of fem mRNA. Nonfunc)onal Fem is made (therefore not shown). Male-­‐specific splicing occurs at Am-­‐dsx due to no regula)on from Fem protein and Dsx protein is made. Male development occurs. (Biewer, et. al 2015; Gempe, et. al 2009; Nissen, et. al 2012) EXPERIMENTAL SUPPORT Figure 6. Am-­‐tra2 gene is essen4al for female splicing of fem and Am-­‐dsx. Nissen et. al (2012) wanted to study the importance of Am-­‐tra2 gene in suppor)ng female development in honeybees. If Am-­‐tra2 was essen)al to female specific splicing of fem and Am-­‐dsx, it was hypothesized that removing Am-­‐tra2 would cause females to show male splicing paierns. Am-­‐tra2 knockdown was induced in embryos by RNA interference. Am-­‐tra2 dsRNA was created from Am-­‐tra2 cDNA previously amplified via RT-­‐PCR from the embryos. This dsRNA was injected into male and female honeybee embryos (4pg, 33pg, or ddH2O) to observe their development. Measurements were taken once bees had developed to larval stage. (A) Removal of Am-­‐tra2 led females to show a male-­‐like splicing paiern (lanes 1-­‐20) compared to controls (lanes 21-­‐30). (B) At the higher concentra)on of 33pg dsRNA-­‐2, females lost splicing ability for fem (lanes 11-­‐20) compared to controls (lanes 26-­‐35). (C) ef-­‐1α is a housekeeping gene used as a control gene. This experiment supports the idea that Am-­‐tra2 is required for sex-­‐specific splicing of fem and Am-­‐dsx mRNA transcripts. (Nissen, et. al 2012) BIG IDEAS •  Sex determined by zygosity of csd gene •  Females require Fem to develop properly •  Am-­‐tra2 promotes female specific splicing of fem and Am-­‐dsx •  Male development results from lack of splicing regula)on FUTURE DIRECTION Figure 2. Honey bee ma4ng. A drone mounts a queen and inserts his endophallus, ejacula)ng semen. Some sperm enters the queen’s spermatheca from which she can ac)vely control which eggs will get fer)lized or not. The queen is ready to lay eggs 3-­‐4 days aVer ma)ng. Drone pulls away from queen, and his endophallus is ripped from his body. This rips open the drone’s abdomen and he quickly dies. •  Inves)ga)on into regula)on of Am-­‐tra2 •  Gene regula)on of Apis mellifera fairly linear, except for Am-­‐tra2 •  Is there more to the mechanism than is known so far? •  Possible experiment: determine regulators of Am-­‐tra2 •  RT-­‐PCR on Am-­‐tra2 mRNA transcripts? •  RNAi experiments on poten)al regulators of Am-­‐tra2? (Orkin) BEES: CRUCIAL TO LIFE REFERENCES Figure 3. Schema4c showing economic contribu4on of bees. (iWonder) Figure 5. Gonadal development in fem-­‐ and csd-­‐repressed honeybee larvae. Gempe, et. al (2009) wanted to see the rela)onship between sex determina)on in the honeybee and fem gene because there had been no previous research on fem involvement in gonad differen)a)on. Genes were repressed using fem and csd siRNAs synthesized using MWG BioTech kits. siRNAs bind to complementary mRNA strands and lead to the mRNA’s degrada)on, effec)vely removing genes from target loca)ons. Gonad differen)a)on is induced early in development, so it was used as an indicator of sex determina)on. Control gonads were taken from untreated honeybees. The csd knockdowns were also used as a control for complete sex change. (A) Normal female gonadal development. (B) Normal haploid male gonadal development. H) When fem was repressed in females, 74% showed development of male testes. (I) In fem-­‐repressed males, no gonadal differences were observed. (J) Females with csd knockdown showed complete male gonadal development with normal testes. (K) Males with csd knockdown showed no change. (Gempe et. al 2009) Biewer, M., Schlesinger F., and M. Hasselmann. (2015). The evolu4onary dynamics of major regulators for sexual development among Hymenoptera species. Front. Gene4. 6(124):1-­‐10 Gempe, T., Hasselmann, M., Schioi, M., Hause, G., Oie, M., and M. Beye. (2009). Sex Determina4on in Honeybees: Two Separate Mechanisms Induce and Maintain the Female Pathway. PLoS Biol. 7(10):
1-­‐10. Honey Bee: Apis mellifera. Retrieved from Na)onal Geographic Society: hip://animals.na)onalgeographic.com/animals/bugs/honeybee/. iWonder: Would we starve without bees? Retrieved from: hip://www.bbc.co.uk/guides/zg4dwmn. Mid-­‐Atlan)c Apiculture Research and Extension Consor)um: Honey Bee Biology. (n.d.) Retrieved from: hips://agdev.anr.udel.edu/maarec/honey-­‐bee-­‐biology/. Nissen, I., Muller, M., and M. Beye. (2012). The Am-­‐tra2 Gene Is an Essen4al Regulator of Female Splice Regula4on at Two Levels of the Sex Determina4on Hierarchy of the Honeybee. Gene4cs 192:1015-­‐1026. Orkin: S)nging Pests, Bees. Retrieved from: hip://www.orkin.com/s)nging-­‐pests/bees/. Schurko, A.M. (2013). To “Bee or Not to Bee” Male or Female? An Educa4onal Primer for Use with “The Am-­‐
tra2 Gene Is an Essen4al Regulator of Female Splice Regula4on at Two Levels of the Sex Determina4on Hierarchy of the Honeybee”. Gene4cs 193:1019-­‐1023. The Biology of the Honey Bee, Apis mellifera. Retrieved from: hip://plantphys.info/plants_human/bees/
bees.shtml. Sex Determination in Clownfish
Brandon Perry
BIOL 354- Developmental Biology
Department of Biological Sciences, Central Washington University, Ellensburg, WA
Clownfish
A. Melanopus is one of 28 different species of clownfish that inhabit the Pacific and Indian oceans, the Red Sea and Australia’s Great Barrier Reef. Also known as anemonefish, they live in symbiosis with sea anemones, cleaning their tentacles and improving water circula)on in exchange for protec)on. Photo by Patzner, R -­‐Clownfish can lay 100-­‐1,000 eggs per clutch -­‐Clownfish are born with both ovarian and tes)cular )ssue -­‐Fully grown clownfish are 2-­‐5 inches long -­‐Average lifespan is 6-­‐10 years in the wild, 3-­‐5 years in cap)vity -­‐Clownfish communicate dominant/subordinate postures by sound Clownfish are easily manipulated into undergoing sex change. This makes them an ideal specimen to study sex change mechanisms in other related, but difficult to study, species. By studying clownfish in their natural habitat, social interac)ons can be observed. Removal of the female is the only necessary task to trigger a sex change event. By removing the females of several different harems and collec)ng the individuals undergoing the subsequent sex change at different stages for lab analysis, a comprehensive framework of social behavior and physiological changes can be made. While the complete sex-­‐determina)on mechanism in clownfish is s)ll unknown, several experiments have been conducted to beier understand the role of endocrine signaling and nuclear receptors. Thyroid hormones and hormone receptors have been shown to be involved in ovary development and in growth and development (Park 2010). Estrogen (E2) and estrogen receptors play a role in ovary matura)on in females, sex change, and gametogenesis in both sexes (Kim 2010). An increase in the stress hormone cor)sol has been observed during the sex change process and linked to the increased aggression that is aiributed to the dominant breeding pair (Godwin 1992). It is also notable that sex change is brought on by a sudden decrease in androgens followed by a surge in E2 and testosterone. Understanding the social interac)ons of clownfish and how they trigger physiological changes is the key to uncovering the mechanism of sex determina)on. Mechanism of Sex Determination
Photo by Patzner, R. Figure 1: Proposed framework of social cues interfacing with physiological changes to drive the sex change mechanism. -­‐  Sex determina)on is linked to social structure -­‐  The exact mechanisms of sex determina)on in clownfish are unknown -­‐  Once triggered, sex change is irreversible -­‐  Gonadal development (key part of sex determina)on)is driven by the presence and absence of hormones Future Directions
-­‐  Can sex change be ar)ficially prevented? -­‐  Hormones that trigger gonadal changes have been iden)fied. Ar)ficially blocking individual hormones/
receptors in the presence of a sex change s)mulus and analyzing target )ssues at different stages may reveal poten)al ac)vator/repressor rela)onships between signals or a )me-­‐con)ngent dosing mechanism. References
Background
-­‐  Clownfish harems consist of a dominant breeding female, a breeding male, and their offspring -­‐  Upon removal of the dominant female, the breeding male undergoes sex change to become the dominant breeding female, and the most aggressive of the offspring undergoes sex determina)on to become the breeding male -­‐  Male to female sex change takes 45-­‐100 days Conclusions
Experimental Design & Results
Colleye O, Parmen)er E (2012) Overview on the Diversity of Sounds Produced by Clownfishes(Pomacentridae): Importance of Acous)c Signals in Their Peculiar Way of Life. PLoS ONE 7(11): e49179 Kim, Na Na et al (2010) Upregula)on of Estrogen Receptor Subtypes and Vitellogenin mRNA in Cinnamon Clownfish Amphiprion melanopus during the sex change process: Profiles on Effects of 17β-­‐Estradiol. Compara)ve Biochemistry and Physiology, Part B Elsevier Inc Park, Mi Seon et al (2010) Molecular Cloning and Expression of TRα and TR β in the Protandrous Cinnamon Clownfish, Amphiprion melanopus during sex reversal. Marine and Freshwater Behaviour and Physiology 43, 371-­‐384 Godwin, J. (1992) The Behavior and Physiology of Protandrous Sex Change In the Cinnamon Anemonefish, Amphiprion melanopus. Diss. U of Hawaii Print Gynandromorph Birds
Chelsea Robbins
BIOL 354- Developmental Biology
Department of Biological Sciences, Central Washington University, Ellensburg, WA
Introduction
Conclusions
Experimental Design & Results
What mechanisms cause Gynandromorphism in chickens? What have we decided? Previous experiments on invertebrates suggest Gynandromorphism is due to the loss of a single sex chromosome. This experiment set out to determine if this was the cause. Three bilateral gynandromorph chickens were obtained and fluorescent in situ hybridiza)on (FISH) analysis with Z and W chromosome probes was used on blood and mul)ple )ssue samples from each side of the birds. The results shows that the blood and )ssue contained a mixture of normal diploid female (ZW) and male (ZZ) cells. This contradicts the implied method of sex chromosome loss and implies a different sex determina)on pathway than known gonadal hormonal influence. This also demonstrates that is very unlikely that this occurs by muta)on and supports the hypothesis that vertebrate gynandromorphs form from failure of the polar body to extrude during meiosis followed by double fer)liza)on. What does it mean? Gynandromorphs are animals that have both male and female parts. A very rare, but naturally occurring phenomenon. Is this phenomenon found in ALL species? No. It has only been found in invertebrates and vertebrate bird species. Recently studied in domes)c chickens (Gallus domes:cus), Zebra finches (Taeniopygia guBata), white-­‐ruffed manakins (Corapipo altera), and house sparrows (Passer domes:cus). How does this occur? Gynandromorphism is believed to occur in invertebrates due to a loss of a single sex cell chromosome but occurs in bird species due to a failure to exclude a polar body during meiosis followed by fer)liza)on of both egg and polar body leading to diploid male and female cell produc)on. Gynandromorphs are oben used to study sex determina)on pathways. How does this affect the animal’s life? Gynandromorph animals are naturally produced. They grow up in the environment normal for their species. No difference has been observed in the animals natural life span although it is believed that they are sterile. No known age of puberty or method of reproduc)on. Most gynandromorphs don’t fit into normal societal standards and therefore are never approached by mates. What mechanism do chickens use to determine sex? Previous experiments on gynandromorph chickens show that sex determina)on cannot be due solely to hormonal influence. This experiment set out to determine if sex phenotype is dependent on cell DNA in contrast to hormones. Transcriptomes of male and female chicken embryos were evalua)on. Researchers found that mRNA of a FAF gene was expressed only in the female and thus must be on the W chromosome. It was also found that a segment of miRNA designated Gallus gallus mir-­‐2954 was expressed 10x higher in the male and most likely found on the Z chromosome. This findings were discovered as early as 18 hours aber incuba)on started long before gonads were developed. This suggests that sex determina)on in chickens is due to cell iden)ty rather than previously thought hormonal influence produced by the gonads. Mechanism of Sex Determination
What is the chicken model for sex determina)on? Sex determina)on in chickens is thought to be dependent only on the iden)ty of the cell and not on hormonal influences. What is the pathway for f orma)on of gynandromorphs? Gynandromorph chickens are thought to occur due to a failure of the polar body to extrude. Male Soma Tes)s Male Phenotype Female Soma Ovaries Female Phenotype Embryo Fer)liza)on Figure 2. Sex determina)on in Chickens. AVer fer)liza)on, cells have inherit female or male iden))es. This leads to gonads forma)on based on cell iden)ty and eventually phenotype of all cells in organism. W Z Z
W
Z ZZ ZW ZZ ZW Figure 3. Sex determina)on in Chicken Gynandromorphs. Polar body fails to extrude during meiosis. Both egg and polar body are fer)lized leading to a normal diploid male/
female gynandromorph. Figure 1. Picture and diagram showing a bilateral gynandromorphic chicken. The right side is female with ZW composi)on and the leV side is male with ZZ composi)on. Gynandromorphs are a natural but rare occurring phenomenon. It has been shown that in invertebrates, this occurrence is due to the loss of a single sex chromosome but vertebrate birds do not fit this criteria. Analysis of Z and W chromosome components using FISH analysis in gynandromorph chickens have shown that they contain normal diploid male/female cells. This suggests that gynandromorph chickens at least are formed due not to a loss of a chromosome but of the failure of the polar body to extrude during meiosis. Further research suggests that sex determinacy in chicken is independently found in the cells themselves and is not dependent solely on hormonal influence. Future Directions
What now? Further ques)ons that need to be answered include: Do other species of birds use similar pathways found in chickens? What causes bilateral Gynandromorphism in contrast to uniform mixtures? Next research project: Further research could include FISH analysis of )ssue from bilateral gynandromorph sparrows to determine if they contain a similar sex determina)on pathway along with analysis of non bilateral gynandromorphs. References
Zhao, D., McBride, D., Nandi, S., McQueen, H. A., McGrew, M. J., Hocking, P.M., Lewis, P.D., Sang, H. M., Clinton, M. (2010). Soma4c sex iden4ty is cell autonomous in the chicken. Nature. 464 (doi: 10.1038/
nature08852). Agate, R. J., Grisham, W., Wade, J., Mann, S., Wingfield, J., Schanen, C., Palo)e, A., Arnold, A. P., (2003), Neural, not gonadal, origin of brain sex differences in a gynandromorphic finch. PNAS. 100 (8). Zhang, S. O., Mathur, S., Haiem, G., Tassy, O., Pourquie, O., (2010). Sex-­‐dimporphic gene expression and ineffec4ve dosage compensa4on of Z-­‐linked genes in gastrula4ng chicken embryos. Biomed Central. 11 (13). Simultaneous Hermaphroditism in Serranus psittacinus
Sierra Wenz
BIOL 354 - Developmental Biology
Department of Biological Sciences, Central Washington University, Ellensburg, WA
Barred Serrano
A Subtropical to tropical sea bass that is na)ve to the eastern pacific. -­‐  Prefers sand or reef terrain -­‐  Grows up to 18 cm -­‐  Refered to as: -­‐  Serranus fasciatus -­‐  Serranus psiiacinus Experimental Design & Results
Ini)al Observa)ons Behavioral observa)ons were made in the summer months between 1981 and 1983 in the Central Gulf of California in the Guaymas -­‐San Carlos area. Researchers observed individuals recording, iden)ty, size, dis)nct markings, and ma)ng behaviors. None of the fish were tagged 25 spawning incidences were observed.Males could be observed frequently in the same loca)on . Hermaphrodites also remained in a home loca)on though it was smaller than the territory of the males. Male territories included hermaphrodite territories but hermaphrodite territories did not overlap. Males spawned in the hermaphrodite home territories and a small percent of hermaphrodites streaked in adjacent hermaphrodite territories (Has)ngs,1986). Conclusions
-­‐  Largest individuals become func)onal males -­‐  There is a posi)ve correla)on between number of eggs and dry ovarian )ssue weight -­‐  There can be a posi)ve correla)on between male reproduc)ve success and tes)s weight. -­‐  This correla)on is not always present depending on the year. -­‐  Ma)ng behaviors are dependent on popula)on density -­‐  No trade off between any of the reproduc)ve parameters measured Future Directions
Serranus psiiacinus (Steen, 2006) Background
-­‐  Simultaneous hermaphrodites -­‐  Have func)oning male and female gonads -­‐  Density dependent ma)ng rituals -­‐  Extremely Low density= Self Fer)liza)on -­‐  Low Density = Long term Monogamy -­‐  Moderate Density= Individual male specializa)on -­‐  High Density= Complex Harems -­‐  Daily egg trading -­‐  Partner releases a clutch of eggs for the other to fer)lize then vice versa -­‐  No parental Care -­‐  External fer)liza)on -­‐  Do they have the ability to reform female reproduc)ve )ssue? Fig. 1: Gonad Morphology of Serranus fasciatus stained -­‐  Add a larger male to the territory of a smaller with Mallory’s triple stain. A. an ovates)s. O indicates male to see if the smaller male reforms ovarian ovarian )ssue T indicates tes)s, ss indicates sperm sinus )ssue and w indicates gonadal wall. C. Male gonad with Fig.2: Male vs Female Reproduc)ve Success (RS). In 1985 regressed Ovarian )ssue (RO) (Has)ngs,1986). -­‐  What causes the change in gonadal alloca)on male RS was posi)vely correlated with male gonad weight, -­‐  Water sample there was no correla)on in 1986. Female RS was determined -­‐  Test for differences in hormone levels in be number of eggs released. The Number of eggs released was posi)vely correlated with the weight of the ovarian )ssue and water the size of the female(Petersen, 1990). -­‐  What genes are involved? -­‐  Use QT PCR to test for differences in gene expression. -­‐  Start with known genes regulated by hormones indicated in the water tests A.) B.) Patterns in Mating Behaviors
-­‐  Determine where in the gonad the genes are expressed with immunohistochemistry References
C.) Figure 2: Ma)ng Tac)cs of Barred Serrano. Represents the ma)ng tac)cs of Barred Serrano under variable popula)on densi)es. Star indicates a func)onal males. A.) Under extremely high popula)on densi)es the largest individual becomes a func)onal male and mates with the largest subordinate hermaphrodites. The largest hermaphrodites protect harems of smaller hermaphrodites whom they mate with as males. The smallest hermaphrodites occasionally act as sneakers and release sperm in a neighboring harem’s ma)ng pair. B.) In Moderate to high popula)on densi)es func)onal males mate with and protect harems of smaller hermaphrodites. The hermaphrodites have a small amount of reproduc)ve success ac)ng as sneakers when the male mates with others in the harem C.) In low popula)on densi)es hermaphrodites monogamously egg swap (Petersen, 2006). Has)ngs, P., & Petersen, C. (1986). A novel sexual paiern in serranid fishes: Simultaneous hermaphrodites and secondary males in Serranus fasciatus. Environ Biol Fish Environmental Biology of Fishes, 59-­‐68. Petersen, C. (1990). Varia4on in reproduc4ve success and gonadal alloca4on in the simultaneous hermaphrodite, Serranus fasciatus. Oecologia, 83(1), 62-­‐67 Petersen, C. (2006). Sexual selec4on and reproduc4ve success in hermaphrodi4c seabasses. Integra)ve and Compara)ve Biology, 46(4), 439-­‐448. Steen R. (Photographer). (2006). Serranus psiiacinus [digital image]. hip://www.discoverlife.org/mp/20q?search=Serranus
+psiiacinus&b=FB14298&l=spanish