Close

Fetal estrogens are not involved in sex determination but critical for early ovarian differentiation in rabbits

Geneviève Jolivet, Nathalie Daniel-Carlier, Erwana Harscoët, Eloïse Airaud, Aurélie Dewaele, Cloé Pierson, Frank Giton, Laurent Boulanger, Nathalie Daniel, Béatrice Mandon-Pépin, Maëlle Pannetier, Eric Pailhoux

Preprint posted on December 14, 2020 https://www.biorxiv.org/content/10.1101/2020.12.13.419770v1.abstract

The lack of fetal aromatase enzyme leads to infertility in rabbit does

Selected by Nándor Lipták

Background

Aromatase enzyme, encoded by the CYP19A1 gene, synthetizes estrogens (estrone, estradiol, and estriol) from androgens. Aromatase deficiency, which is caused by mutations in the CYP19A1 gene, may evoke primary amenorrhea, pseudo hermaphroditism, virilization in humans (OMIM: 613546, (Jones et al. 2007)). The first peak of aromatase expression usually starts during embryogenesis, around implantation. The second peak occurs during fetal life in the ovaries in rabbits and several other mammalian species (e.g. sheeps, bovines, humans, etc.), but not in rodents.

Due to the aforementioned differences in aromatase expression between humans and rodents, the authors created CYP19A1 knockout (KO) rabbits to examine the consequences of the lack of fetal aromatase. Thus, the aim of this preprint was to investigate the role of the fetal peak of estrogen in ovary development and differentiation.

 

Key findings

CYP19A1 KO embryos were created by the microinjections of one-cell stage embryos with equimolar mixture of the left and right arm TALEN mRNAs (50ng/μl each). Injected embryos were transferred to recipient female rabbits (does) using laparoscopic surgery. Three mutant rabbit lines, harboring 339, 498 and 829 base-pairs deletions, respectively were established.

No estradiol in the serum of ARO-/- XX rabbits

Estradiol was not detected in 28 days old ARO-/- XX fetuses. Estradiol in the serum of ARO-/- does were undetectable, while serum estradiol concentrations were increased with age in WT and ARO+/- heterozygous does. Serum testosterone concentrations were similar in case of all genotypes.

ARO+/- heterozygous rabbits from both sexes are fertile, while ARO-/- does are infertile

The fertility of heterozygous rabbits was normal, but ARO-/- does had small genital tracts, and their ovaries had almost no primordial follicles, primary and secondary follicles. Histological analysis revealed the lack of ovulation rupture points in ARO-/- does after hormonal treatment.

The lack of aromatase did not disturb the sex determination of the early fetal gonad

 

Estrogens are involved in early ovarian development

From the 20th day post coitum, ARO-/- fetuses had thinner ovary cortex compared with the ARO+/- fetuses. In addition, the number of germ cells and the proliferation of the somatic cells in the coelomic epithelium were reduced.

Why I liked this preprint

The vast majority of research groups use CRISPR/Cas9 technology to create KO rabbits. In this preprint, three KO rabbit lines were created with the TALEN method, proving the high efficiency of this KO technology in rabbits. The results of this preprint may have good translational value regarding the aromatase deficiency-induced infertility in women.

Questions for the authors

  1. Estrogen replacement was successful to attenuate the deleterious effects of aromatase deficiency, both in men and women (Mullis et al. 1997; Herrmann et al. 2002; Jones et al. 2007). Have you made any attempt to rescue the infertile phenotype of the ARO-/- does?
  2. According to the Materials and Methods section, superovulation was induced in the donor does by five injections of follicle-stimulating hormone (FSH), then one injection of human chorionic gonadotropin (hCG) (Peyny et al. 2020). But there is another accepted method for superovulation in rabbits, the pregnant mare’s serum gonadotropin PMSG/hCG protocol (Besenfelder & Brem 1993), which includes only two hormonal injections, and therefore causes minimal suffering for the donor does. What was the reason of choosing the more complicated and more painful FSH/hCG protocol?
  3. In the majority of the case reports, aromatase deficiency is caused by heterozygous mutations in human patients. Are you planning any further characterization of the ARO+/- rabbit lines?
  4. Homozygous mutations in the CYP19A1 gene were reported in boys and in an adult man (Deladoey et al. 1999; Herrmann et al. 2002). In your preprint, ARO+/- heterozygous bucks, does, and ARO-/- homozygous does were characterized in detail. Did the ARO-/- bucks have any different symptoms or phenotype compared with the symptoms of the aforementioned genotypes?
  5. Aromatase deficiency is usually evoked by deletions, insertions or substitutions of 1-3 base pair(s) in the CYP19A1 gene in human patients. Would it be more advantageous to use base-editing systems instead of TALEN to create CYP19A1 mutant rabbits?

 

References

Besenfelder U. & Brem G. (1993) Laparoscopic Embryo-Transfer in Rabbits. Journal of Reproduction and Fertility 99, 53-6.

Deladoey J., Fluck C., Bex M., Yoshimura N., Harada N. & Mullis P.E. (1999) Aromatase deficiency caused by a novel P450arom gene mutation: impact of absent estrogen production on serum gonadotropin concentration in a boy. J Clin Endocrinol Metab 84, 4050-4.

Herrmann B.L., Saller B., Janssen O.E., Gocke P., Bockisch A., Sperling H., Mann K. & Broecker M. (2002) Impact of estrogen replacement therapy in a male with congenital aromatase deficiency caused by a novel mutation in the CYP19 gene. J Clin Endocrinol Metab 87, 5476-84.

Jones M.E., Boon W.C., McInnes K., Maffei L., Carani C. & Simpson E.R. (2007) Recognizing rare disorders: aromatase deficiency. Nat Clin Pract Endocrinol Metab 3, 414-21.

Mullis P.E., Yoshimura N., Kuhlmann B., Lippuner K., Jaeger P. & Harada H. (1997) Aromatase deficiency in a female who is compound heterozygote for two new point mutations in the P450arom gene: impact of estrogens on hypergonadotropic hypogonadism, multicystic ovaries, and bone densitometry in childhood. J Clin Endocrinol Metab 82, 1739-45.

Peyny M., Jarrier-Gaillard P., Boulanger L., Daniel N., Lavillatte S., Cadoret V., Papillier P., Monniaux D., Peynot N., Duranthon V., Jolivet G. & Dalbies-Tran R. (2020) Investigating the role of BCAR4 in ovarian physiology and female fertility by genome editing in rabbit. Sci Rep 10, 4992.

 

Posted on: 25th January 2021 , updated on: 23rd June 2021

doi: https://doi.org/10.1242/prelights.27119

Read preprint (No Ratings Yet)




Author's response

Dr. Geneviève Jolivet shared

Q1: Estrogen replacement was successful to attenuate the deleterious effects of aromatase deficiency, both in men and women (Mullis et al. 1997; Herrmann et al. 2002; Jones et al. 2007). Have you made any attempt to rescue the infertile phenotype of the ARO-/- does?

Answer: No, we did not make any attempt to rescue the infertile phenotype through estrogen replacements, or any other treatment. In the human, estrogen replacements are given after birth, in the young girl when external deleterious effects are detected and modify the development of the genital tract, or breast, or in boys at around puberty when metabolic disorders appear.

In our study, estrogen replacement could have been given during early pregnancy to compensate the lack of estrogen in the fetal ovary. However, it would have been necessary to treat individually each fetus in utero. Indeed, we have observed that the gonads of KO rabbit fetuses were void of estrogens or androgens, thus showing that the maternal steroids do not reach the fetal gonad. Giving treatment to the mother would have no effect. Moreover, estrogens probably act in the fetal gonads through autocrine or paracrine interactions. Thus, in spite of the technical possibility to inject each fetus through ultrasound monitoring, we have no insurance that the injected estrogen would enter inside the gonad and target cells in a proper manner.

Q2: According to the Materials and Methods section, superovulation was induced in the donor does by five injections of follicle-stimulating hormone (FSH), then one injection of human chorionic gonadotropin (hCG) (Peyny et al. 2020). But there is another accepted method for superovulation in rabbits, the pregnant mare’s serum gonadotropin PMSG/hCG protocol (Besenfelder & Brem 1993), which includes only two hormonal injections, and therefore causes minimal suffering for the donor does. What was the reason of choosing the more complicated and more painful FSH/hCG protocol?

Answer: By the past, we have attempted to switch the 5-injections protocol to the 2-injections one as you propose. Indeed this latter was tempting. However, in our rabbit strain, the 2-injections protocol was far less efficient and resulted in a poor number of embryos. Anyway, the pain is more the consequence of the growing of follicles than of the number of injections. An efficient method for superovulation is basically painful, as many women who are treated with hormones during assisted reproduction say. Thus, we preferred to use the most efficient method to get the largest number of embryos from the smallest number of does.

Q3: In the majority of the case reports, aromatase deficiency is caused by heterozygous mutations in human patients. Are you planning any further characterization of the ARO+/- rabbit lines?

Answer: In the human, there is no consistent ovarian phenotype in aromatase deficiency specifically when mutations are heterozygous. In the ARO+/- rabbits, we did not detect any clear phenotype. Thus, the further characterization of these rabbits is not a priority.

Q4: Homozygous mutations in the CYP19A1 gene were reported in boys and in an adult man (Deladoey et al. 1999; Herrmann et al. 2002). In your preprint, ARO+/- heterozygous bucks, does, and ARO-/- homozygous does were characterized in detail. Did the ARO-/- bucks have any different symptoms or phenotype compared with the symptoms of the aforementioned genotypes?

Answer: To date, the ARO-/- bucks are fertile and are used for reproduction. However, the description of these males is under study in our laboratory and is to be published soon.

Q5: Aromatase deficiency is usually evoked by deletions, insertions or substitutions of 1-3 base pair(s) in the CYP19A1 gene in human patients. Would it be more advantageous to use base-editing systems instead of TALEN to create CYP19A1 mutant rabbits?

Answer: Our aim was to analyze the effect of the total absence of aromatase in order to investigate the role of the early fetal expression of aromatase in the ovary. By using base editing systems, we could of course more specifically study the impact caused by mutations already identified in humans. That is not the same purpose. Indeed, one could imagine that aromatase mutants have specific enzymatic effects responsible for specific phenotypes.

Have your say

Your email address will not be published. Required fields are marked *

This site uses Akismet to reduce spam. Learn how your comment data is processed.

Sign up to customise the site to your preferences and to receive alerts

Register here

preLists in the developmental biology category:

2nd Conference of the Visegrád Group Society for Developmental Biology

Preprints from the 2nd Conference of the Visegrád Group Society for Developmental Biology (2-5 September, 2021, Szeged, Hungary)

 



List by Nándor Lipták

Fibroblasts

The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!

 



List by Osvaldo Contreras

EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)

A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.

 



List by Alex Eve

EMBL Conference: From functional genomics to systems biology

Preprints presented at the virtual EMBL conference "from functional genomics and systems biology", 16-19 November 2020

 



List by Jesus Victorino

Single Cell Biology 2020

A list of preprints mentioned at the Wellcome Genome Campus Single Cell Biology 2020 meeting.

 



List by Alex Eve

Society for Developmental Biology 79th Annual Meeting

Preprints at SDB 2020

 



List by Irepan Salvador-Martinez, Martin Estermann

FENS 2020

A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020

 



List by Ana Dorrego-Rivas

Planar Cell Polarity – PCP

This preList contains preprints about the latest findings on Planar Cell Polarity (PCP) in various model organisms at the molecular, cellular and tissue levels.

 



List by Ana Dorrego-Rivas

Cell Polarity

Recent research from the field of cell polarity is summarized in this list of preprints. It comprises of studies focusing on various forms of cell polarity ranging from epithelial polarity, planar cell polarity to front-to-rear polarity.

 



List by Yamini Ravichandran

TAGC 2020

Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20

 



List by Maiko Kitaoka et al.

3D Gastruloids

A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells). Updated until July 2021.

 



List by Paul Gerald L. Sanchez and Stefano Vianello

ASCB EMBO Annual Meeting 2019

A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)

 



List by Madhuja Samaddar et al.

EDBC Alicante 2019

Preprints presented at the European Developmental Biology Congress (EDBC) in Alicante, October 23-26 2019.

 



List by Sergio Menchero et al.

EMBL Seeing is Believing – Imaging the Molecular Processes of Life

Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019

 



List by Dey Lab

SDB 78th Annual Meeting 2019

A curation of the preprints presented at the SDB meeting in Boston, July 26-30 2019. The preList will be updated throughout the duration of the meeting.

 



List by Alex Eve

Lung Disease and Regeneration

This preprint list compiles highlights from the field of lung biology.

 



List by Rob Hynds

Young Embryologist Network Conference 2019

Preprints presented at the Young Embryologist Network 2019 conference, 13 May, The Francis Crick Institute, London

 



List by Alex Eve

Pattern formation during development

The aim of this preList is to integrate results about the mechanisms that govern patterning during development, from genes implicated in the processes to theoritical models of pattern formation in nature.

 



List by Alexa Sadier

BSCB/BSDB Annual Meeting 2019

Preprints presented at the BSCB/BSDB Annual Meeting 2019

 



List by Dey Lab

Zebrafish immunology

A compilation of cutting-edge research that uses the zebrafish as a model system to elucidate novel immunological mechanisms in health and disease.

 



List by Shikha Nayar
Close