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

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.

 

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

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