Differentiation 91 (2016) 78–89 Contents lists available at ScienceDirect Differentiation journal homepage: www.elsevier.com/locate/diff Review article Investigation of sexual dimorphisms through mouse models and hormone/hormone-disruptor treatments Lerrie Ann Ipulan a,1, Dennis Raga a,1, Kentaro Suzuki a, Aki Murashima a, Daisuke Matsumaru a, Gerald Cunha b, Gen Yamada a,n a Department of Developmental Genetics, Institute of Advanced Medicine, Wakayama Medical University (WMU), Wakayama, Japan b Department of Urology, School of Medicine, University of California San Francisco, San Francisco, CA, USA article info abstract Article history: Sexual dimorphism in mouse reproductive tissues is observable in adult, post-natal, and embryonic Received 8 November 2015 stages. The development of sexually dimorphic tissues starts with an ambisexual structure. It is followed Accepted 11 November 2015 by sex-specific organogenesis as guided by different signaling pathways that occur from late embryonic Available online 2 December 2015 stages. The measurement of the anogenital distance (AGD), and the observation of the external genitalia Keywords: are practical ways to distinguish male and female pups at birth and thereafter. Careful observation of the Reproductive tissue morphological or histological features and the molecular signatures of the external genitalia and peri- Sexual dimorphism neum enable identification of sex or feminization/masculinization of embryos. Aberrations in hormone Mouse Cre lines signaling via castration or treatment with hormones or hormone disruptors result in dysmorphogenesis Hormone of reproductive tissues. Several hormone disruptors have been used to modulate different aspects of Hormone disruption hormone action through competitive inhibition and exogenous hormone treatment. Concomitantly, the Androgen Perineum vast advancement of conditional mutant mouse analysis leads to the frequent utilization of Cre re- External genitalia combination technology in the study of reproductive/urogenital tissue development. Mouse Cre-lines that are tissue-specific and cell-specific are also effective tools in identifying the molecular mechanisms during sexually dimorphic development. Cre-lines applicable to different cell populations in the prostate, seminal vesicles, testis and ovaries, and mammary glands are currently being utilized. In the external genitalia and perineum, Cre lines that examine the signaling pathways of cells of endodermal, ecto- dermal, and mesenchymal origin reveal the roles of these tissues in the development of the external genitalia. The interaction of hormones and growth factors can be examined further through a variety of techniques available for researchers. Such cumulative information about various technologies is sum- marized. & 2015 International Society of Differentiation. Published by Elsevier B.V. All rights reserved. Contents 1. Introduction . 79 2. Sexual dimorphism in mouse reproductive tissues . 79 2.1. Sexual dimorphism of external reproductive tissues at post-natal stages . 80 2.2. Sexual dimorphism of external reproductive tissues at embryonic stages . 80 3. Experimental techniques in hormonal modulation and sexual dimorphism . 81 3.1. Cessation of androgen signaling by gonadectomy and hormone inhibition experiments . 81 3.2. Hormonal modulators are essential tools to determine the mechanisms of sexual differentiation in model animals . 82 3.3. Critical time window for hormonal modulation and sexual dimorphism. 82 3.4. Usefulness of animal models and application to human studies: variations and limitations . 82 4. Transgenic mouse lines for investigating reproductive structures . 82 4.1. The use of transgenic mouse lines for the investigation of external genitalia and perineum . 84 n Corresponding author at: Department of Developmental Genetics, Institute of Advanced Medicine, Wakayama Medical University (WMU), Wakayama, Japan. E-mail addresses: [email protected], [email protected] (G. Yamada). 1 Equal contributing author. http://dx.doi.org/10.1016/j.diff.2015.11.001 Join the International Society for Differentiation (www.isdifferentiation.org) 0301-4681/& 2015 International Society of Differentiation. Published by Elsevier B.V. All rights reserved. L.A. Ipulan et al. / Differentiation 91 (2016) 78–89 79 4.1.1. GT outgrowth abnormalities . 84 4.1.2. Hypospadias-like phenotype and ventral GT hypoplasia . 84 4.1.3. Perineum Defects. 87 5. Conclusion . 87 Disclosure statement. 87 Acknowledgment . 87 References..............................................................................................................87 1. Introduction Information about different hormone and hormone-disruptors is also discussed together with technical tips in utilizing such che- The reproductive system is essential for the continuity of spe- micals and their interpretation. cies. The differences and the complimentary nature of male and female reproductive systems provide optimal fertility, ensure re- production, and facilitate the continuous caring for the offspring. 2. Sexual dimorphism in mouse reproductive tissues The female reproductive system is composed of ovaries, oviducts, uterus, cervix and vagina. Other sexual characteristics include The development of reproductive tissues starts with bipotential mammary glands and fat deposition in the torso. The male re- primordial organ formation. It is followed by a sexually dimorphic productive system is composed of testes, epididymes, vas de- developmental stage, which is governed by molecular pathways ferens, urethra, seminal vesicles, prostate gland, bulbourethral leading to morphologically different male and female structures. glands, and penis. Fig. 1 summarizes the timeline of the bipotential stage, the stage of The study of reproductive tract tissues requires a multi- molecular sexual differences, and the stage of observable mor- disciplinary approach. The morphological changes and effects of phological dimorphism of reproductive tract organs. The onset of hormone balance on developing male and female reproductive molecular sexual differences refers to the stage wherein differ- tracts distinguish them from other organ systems. Disruptions in ences in signaling or gene expression in such tissues can be de- hormone signaling affect both the morphology and subsequent tected, which is clearly a moving target subject to continued im- adult physiological functions of reproductive tract tissues. As such, provement in analytical techniques. Some detailed histological factors related with stage-dependent perturbations, tissue-speci- differences and cellular differentiation may occur concomitant ficity, and proper mouse models in the development of such tis- with molecular sexual differences or shortly thereafter. Morpho- sues must be considered. logical dimorphism is defined as a stage wherein structural dif- This review aims to present technical information for the in- ferences are apparent. vestigation of male and female reproductive tracts such as ob- The ovary and the testis, which originate from the genital ridge, servable sexual dimorphisms, timeline of development, and ap- are the first structures to undergo molecular sexual differentiation propriate mouse Cre lines for conditional mutagenesis (with par- (Koopman et al., 1991; Wilhelm and Koopman, 2006). The testes ticular focus on external genitalia and perineum formation). then secrete hormones (androgens, anti-Mullerian hormone) that Fig. 1. Timeline of development and sexual dimorphisms of reproductive structures. This figure summarizes the approximate timepoints in the development of reproductive structures, which are divided into (i) the bipotential stage, the phase of ambisexual organ formation; (ii) the stage of initiation of molecular sexual differences, the phase of observed differences in signaling pathways/gene expression patterns with subtle histological differences; and (iii) the stage of morphological sexual dimorphisms. (AGD – anogenital distance, BC – bulbocavernosus). 80 L.A. Ipulan et al. / Differentiation 91 (2016) 78–89 initiate or regulate male accessory organ development (Franco and 2.1. Sexual dimorphism of external reproductive tissues at post-natal Yao, 2012). This implies the essential role of hormones in sexually stages dimorphic development. The cloaca divides into the urogenital si- nus (UGS) and rectal/anal compartments. After the differentiation of Mice at early post-natal stages can be sexed according to the the gonads, the Wolffian ducts (WD) and Mullerian ducts (MD) AGD (Fig. 2c–d), inguinal mammary teats, and the small darkened form within the urogenital ridge at embryonic day 13.5 (E13.5) in region in the perineum (Fig. 2c–d), which is applicable for non- both male and female embryos (Kobayashi et al., 2011; Murashima albino mice (Schneider et al., 1978). All other developmental et al., 2015, 2011). In male embryos, the Mullerian ducts degenerate characteristics in the early post-natal stage such as weight gain, as a result of action of anti-Mullerian hormone, while in female lanugo, hair growth, incisor eruption, and opening of the eyelids embryos the Wolffian ducts degenerates as a consequence of the showed non-sex-biased distinctions (Greenham and Greenham, absence of androgens. After E16.0 the MDs of females form the 1977). The AGD of female albino pups have a range between oviduct, uterine horns, cervical canal and upper vagina in female. 1.0 and 2.1 mm with a 1.6 mm mean, while male pups have an The lower vagina or sinus vagina is formed from the