Freemartin in the amphibian Pleurodeles waltl: Parabiosis between individuals from opposite sex triggers both germ and somatic cells alterations during female gonad development

Abstract

Wild type embryos of the newt Pleurodeles waltl were used to realize parabiosis, a useful model to study the effect of endogenous circulating hormones on gonad development. The genotypic sex of each parabiont (ZZ male or ZW female) was determined early from the analysis of the sex chromosome borne marker peptidase-1. In ZZ/ZZ and ZW/ZW associations, gonads develop according to genetic sex. In ZZ/ZW associations, the ZZ gonads differentiate as normal testes while ZW gonads development shows numerous alterations. At the beginning of sex differentiation, these ZW gonads possess a reduced number of germ cells and a reduced expression of steroidogenic factor 1 and P450-aromatase mRNAs when compared to gonads from ZW/ZW associations. During gonad differentiation, conversely to the control situation, these germ cells do not enter meiosis as corroborated by chromatin status and absence of the meiosis entry marker DMC1; the activity of the estradiol-producing enzyme P450-aromatase is as low as in ZZ gonads. At adulthood, no germ cells are observed on histological sections, consistently with the absence of VASA expression. At this stage, the testis-specific marker DMRT1 is expressed only in ZZ gonads, suggesting that the somatic compartment of the ZW gonad is not masculinized. So, when exposed to ZZ hormones, ZW gonads reach the undifferentiated status but the ovary differentiation does not occur. This gonad is inhibited by a process affecting both somatic and germ cells. Additionally, the ZW gonad inhibition does not occur in the case of an exogenous estradiol treatment of larvae.

Evidence for a conserved role of retinoic acid in urodele amphibian meiosis onset

“…The amplification was performed with 0.5 U of Taq DNA Polymerase (Invitrogen) in PCR buffer containing 25 mM of each deoxynucleotide triphosphate, 1.5 mM MgCl 2 , and 10 pmol of each primer in a total volume of 25 l. Specific primers and conditions of amplification for PwGapdh, PwVasa, and PwDmc1 cDNAs have been described previously (Dumond et al, 2008b). Primers for PwCyp26b1 (dir 5Ј-GAC-AAG-AGC-TGC-AAG-CTG-CC-3Ј; rev 5Ј-GCT-GGA-TCT-TGG-GCA-GGT-A-AC-3Ј), PwRaldh2 (dir 5Ј-CCC-ATT-GGA-GTG-TGT-GGA-CAG-ATC-3Ј; rev 5Ј-CTT -CTT-CCA-GCT-GCT-TCT-TG-3Ј), and PwSox9 (GenBank accession no.…”

“…Each cDNA sample was analyzed in triplicate with specific PwVasa or PwDmc1 primers (Dumond et al, 2008b). PwDmc1 expression was normalized to PwVasa transcript expression as a germ cell control gene.…”

“…GM complexes were fixed in Bouin's solution, embedded in paraffin, and sectioned at 7 m. Sections were stained with hematoxylin-eosin-light green as described previously (Dumond et al, 2008b).…”

“…To determine at which developmental stage meiosis entry takes place in standard male (ZZ) and female (ZW) animals, we first analyzed the expression of the meiosis marker PwDmc1 (Dumond et al, 2008b). Then, we designed a protocol for organotypic cultures of GM complexes to evaluate the involvement of retinoid and steroid hormones in meiosis initiation.…”

Sexual dimorphism of AMH, DMRT1 and RSPO1 localization in the developing gonads of six anuran species

“…Moreover, AMH is expressed in the granulosa cells in the ovarian follicles, inhibiting the growth of the latter (Vigier et al, 1984). The expression pattern of AMH has never been described in amphibians (Dumond et al, 2008).…”