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Nadine M Richings Department of Zoology, The University of Melbourne, Victoria, 3010, Australia

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Geoffrey Shaw Department of Zoology, The University of Melbourne, Victoria, 3010, Australia

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Peter D Temple-Smith Department of Zoology, The University of Melbourne, Victoria, 3010, Australia

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Marilyn B Renfree Department of Zoology, The University of Melbourne, Victoria, 3010, Australia

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Here we report the first use of intra-cytoplasmic sperm injection (ICSI) in a marsupial, the tammar wallaby (Macropus eugenii ), to achieve in vitro fertilization and cleavage. A single epididymal spermatozoon was injected into the cytoplasm of each mature oocyte collected from Graafian follicles or from the oviduct within hours of ovulation. The day after sperm injection, oocytes were assessed for the presence of pronuclei and polar body extrusion and in vitro development was monitored for up to 4 days. After ICSI, three of four (75%) follicular and four of eight (50%) tubal oocytes underwent cleavage. The cleavage pattern was similar to that previously reported for in vivo fertilized oocytes placed in culture, where development also halted at the 4- to 8-cell stage. One-third of injected oocytes completed the second cleavage division, but only a single embryo reached the 8-cell stage. The success of ICSI in the tammar wallaby provided an opportunity to examine the influence of the mucoid coat that is deposited around oocytes passing through the oviduct after fertilization. The presence of a mucoid coat in tubal oocytes did not prevent fertilization by ICSI and the oocytes cleaved in vitro to a similar stage as follicular oocytes lacking a mucoid coat. Cell–zona and cell–cell adhesion occurred in embryos from follicular oocytes, suggesting that the mucoid coat is not essential for these processes. However, blastomeres were more closely apposed in embryos from tubal oocytes and cell–cell adhesion was more pronounced, indicating that the mucoid coat may be involved in maintaining the integrity of the conceptus during cleavage.

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Yu Chen School of BioSciences, The University of Melbourne, Victoria, Australia

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Jaiden Lay School of BioSciences, The University of Melbourne, Victoria, Australia

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Geoffrey Shaw School of BioSciences, The University of Melbourne, Victoria, Australia

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Gerard A Tarulli School of BioSciences, The University of Melbourne, Victoria, Australia

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Marilyn B Renfree School of BioSciences, The University of Melbourne, Victoria, Australia

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In brief

Atrazine, like oestrogen, disorganises laminin formation and reduces the number of germ cells and Sertoli cells in the developing testes of the tammar wallaby. This study suggests that interfering with the balance of androgen and oestrogen affects the integrity of laminin structure and testis differentiation.

Abstract

The herbicide atrazine was banned in Europe in 2003 due to its endocrine disrupting activity but remains widely used. The integrity of the laminin structure in fetal testis cords requires oestrogen signalling but overexposure to xenoestrogens in the adult can cause testicular dysgenesis. However, whether xenoestrogens affect laminin formation in developing testes has not been investigated. Here we examined the effects of atrazine in the marsupial tammar wallaby during early development and compare it with the effects of the anti-androgen flutamide, oestrogen, and the oestrogen degrader fulvestrant. The tammar, like all marsupials, gives birth to altricial young, allowing direct treatment of the developing young during the male programming window (day 20–40 post partum (pp)). Male pouch young were treated orally with atrazine (5 mg/kg), flutamide (10 mg/kg), 17β-oestradiol (2.5 mg/kg) and fulvestrant (1 mg/kg) daily from day 20 to 40 pp. Distribution of laminin, vimentin, SOX9 and DDX4, cell proliferation and mRNA expression of SRY, SOX9, AMH, and SF1 were examined in testes at day 50 post partum after the treatment. Direct exposure to atrazine, flutamide, 17β-oestradiol, and fulvestrant all disorganised laminin but had no effect on vimentin distribution in testes. Atrazine reduced the number of germ cells and Sertoli cells when examined at day 40–50 pp and day 20 to 40 pp, respectively. Both flutamide and fulvestrant reduced the number of germ cells and Sertoli cells. Atrazine also downregulated SRY expression and impaired SOX9 nuclear translocation. Our results demonstrate that atrazine can compromise normal testicular differentiation during the critical male programming window.

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Natalie E Calatayud Department of Zoology, The University of Melbourne, Melbourne, Victoria 3010, Australia

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Andrew J Pask Department of Zoology, The University of Melbourne, Melbourne, Victoria 3010, Australia

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Geoffrey Shaw Department of Zoology, The University of Melbourne, Melbourne, Victoria 3010, Australia

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Nadine M Richings Department of Zoology, The University of Melbourne, Melbourne, Victoria 3010, Australia

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Sue Osborn Department of Zoology, The University of Melbourne, Melbourne, Victoria 3010, Australia

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Marilyn B Renfree Department of Zoology, The University of Melbourne, Melbourne, Victoria 3010, Australia

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Oestrogen has wide ranging effects in development mediated mainly via the two oestrogen receptors, α (ESR1, also known as ERα) and β (ESR2, also known as ERβ). Oestrogen is the key factor that directs the indifferent gonad to become an ovary in many non-mammalian vertebrates. Oestrogen is not required for early ovarian differentiation in mammals but can disrupt normal testicular development in eutherians. Surprisingly, exogenous oestrogen can cause sex reversal of an XY gonad in two marsupials, the North American opossum and the tammar wallaby. To understand the mechanism by which oestrogen induces sex reversal, we characterised the genes for ESR1 and ESR2 and examined their expression during gonadal differentiation in the tammar wallaby, Macropus eugenii. Both receptors were expressed in the somatic cells and germ cells of the indifferent gonad in both XX and XY foetuses throughout all stages of development, and persisted in these cells into adulthood. ERs were also present in many other tissues including kidney, pituitary and mammary gland. ER mRNA was not significantly altered by exogenous oestrogen in cultured XY gonads but the receptors translocated to the nucleus in its presence. These findings confirm that there is conserved expression of the ERs in the indifferent gonad despite the lack of available ligand during early gonadal development. The receptors can respond to exogenous estrogen at this early stage and are capable of transducing signals in the early mammalian gonad. However, the selective forces that maintained conserved ER expression in this tissue remain unknown.

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