pubmed.ncbi.nlm.nih.gov

CYP26 Enzymes Are Necessary Within the Postnatal Seminiferous Epithelium for Normal Murine Spermatogenesis - PubMed

CYP26 Enzymes Are Necessary Within the Postnatal Seminiferous Epithelium for Normal Murine Spermatogenesis

Cathryn A Hogarth et al. Biol Reprod. 2015 Jul.

Abstract

The active metabolite of vitamin A, retinoic acid (RA), is known to be essential for spermatogenesis. Changes to RA levels within the seminiferous epithelium can alter the development of male germ cells, including blocking their differentiation completely. Excess RA has been shown to cause germ cell death in both neonatal and adult animals, yet the cells capable of degrading RA within the testis have yet to be investigated. One previous study alluded to a requirement for one of the RA degrading enzymes, CYP26B1, in Sertoli cells but no data exist to determine whether germ cells possess the ability to degrade RA. To bridge this gap, the roles of CYP26A1 and CYP26B1 within the seminiferous epithelium were investigated by creating single and dual conditional knockouts of these enzymes in either Sertoli or germ cells. Analysis of these knockout models revealed that deletion of both Cyp26a1 and Cyp26b1 in either cell type resulted in increased vacuolization within the seminiferous tubules, delayed spermatid release, and an increase in the number of STRA8-positive spermatogonia, but spermatozoa were still produced and the animals were found to be fertile. However, elimination of CYP26B1 activity within both germ and Sertoli cells resulted in severe male subfertility, with a loss of advanced germ cells from the seminiferous epithelium. These data indicate that CYP26 activity within either Sertoli or germ cells is essential for the normal progression of spermatogenesis and that its loss can result in reduced male fertility.

Keywords: CYP26; Sertoli cells; retinoic acid; spermatogenesis; testis.

© 2015 by the Society for the Study of Reproduction, Inc.

PubMed Disclaimer

Figures

FIG. 1
FIG. 1

CYP26A1 activity is not essential for normal spermatogenesis within the seminiferous epithelium. Images show representative cross-sections of testes from the three different lines of Cyp26a1 Flox/+; Cre control and Flox/Flox; Cre mutant male mice 15 (A–D) and 90 (E–H) dpp, and stained for STRA8 protein via immunohistochemistry (brown precipitate). The Stra8-iCre line is shown in B and F, the Amh-Cre line is shown in C and G, and the dual Cre line is shown in D and H. A representative cross-section of an epididymis collected from a Flox/Flox; dual Cre animal is shown in the inset (H). Flox/+;Cre control animals were collected for all three different Cre lines, but as they showed identical morphology, only one representative image was selected for presentation (A and E). Black arrows denote STRA8-positive preleptotene spermatocytes, and red arrows indicate STRA8-positive spermatogonia. Bars = 100 μm.

FIG. 2
FIG. 2

Mild spermatogenic defects resulted following a loss of CYP26B1 activity within Sertoli cells. Images show representative cross-sections of testes from two different lines of Cyp26b1 Flox/+; Cre control and Flox/Flox; Cre mutant male mice 15 (A–C) and 90 (D–F) dpp and stained for STRA8 protein via immunohistochemistry (brown precipitate). The Stra8-iCre line is shown in B and E, and the Amh-Cre line is shown in C and F. Flox/+;Cre control animals were collected for both Cre lines, but as they showed identical morphology, only one representative image was selected for presentation (A and D). Black arrows denote STRA8-positive preleptotene spermatocytes, red arrows indicate STRA8-positive spermatogonia, and asterisks indicate either degraded tubules or cross-sections missing advanced germ cells. Bars = 100 μm.

FIG. 3
FIG. 3

Elimination of both CYP26A1 and CYP26B1 activity from either germ or Sertoli cells induces mild spermatogenic defects. A–I) Representative cross-sections of testes from the two different lines of Cyp26a1/b1 Flox/+; Cre control and Flox/Flox; Cre mutant male mice 15 (A–C), 30 (D–F), and 90 (G–I) dpp and stained for STRA8 protein via immunohistochemistry (brown precipitate). The Stra8-iCre line is shown in B, E, and H, and the Amh-Cre line is shown in C, F, and I. Flox/+;Cre control animals were collected for both Cre lines but as they showed identical morphology, only one representative image was selected for presentation (A, D, and G). Black arrows denote STRA8-positive preleptotene spermatocytes, red arrows designate STRA8-positive spermatogonia, green arrows point to vacuoles formed within the seminiferous epithelium, blue arrows highlight sloughed advanced germ cells, and asterisks indicate cross-sections missing advanced germ cells. Bars = 100 μm. J) Graph shows the testis:body weight ratio (Y axis), measured in grams, for both of the Cre lines. Error bars represent SEM. K) Graph shows the synchronization factor (Y axis) calculated for both of the Cre lines. J and K) Errors bars are SEM.

FIG. 4
FIG. 4

Impaired spermatid release occurs in the Cyp26a1/b1-single Cre conditional mutants. Images show representative cross-sections of stage VIII testis tubules from the two different lines of Cyp26a1/b1 Flox/+; Cre control and Flox/Flox; Cre mutant male mice 90 dpp and stained for STRA8 protein via immunohistochemistry (brown precipitate) (A–C). The Stra8-iCre line is shown in B, and the Amh-Cre line is shown in C. Flox/+;Cre control animals were collected for both of the Cre lines, but as they showed identical morphology, only one representative image was selected for presentation (A). Black arrows denote STRA8-positive preleptotene spermatocytes, and red arrows indicate elongated spermatids embedded within the seminiferous epithelium at stage VIII of the cycle. Bars = 50 μm.

FIG. 5
FIG. 5

STRA8 is prematurely expressed by spermatogonia within Cyp26a1/b1-single Cre conditional mutants. A–F) Representative cross-sections of stage II to VI testis tubules from the two different lines of Cyp26a1/b1 Flox/+; Cre control and Flox/Flox; Cre mutant male mice aged 90 dpp and stained for either STRA8 (A–C) or ZBTB16 (D–F) protein via immunohistochemistry (brown precipitate). The Stra8-iCre line is shown in B and E, and the Amh-Cre line is shown in C and F. Flox/+;Cre control animals were collected for both of the Cre lines, but as they showed identical staining patterns, only one representative image was selected for presentation (A and D). Black arrows denote ZBTB16-positive spermatogonia, and red arrows designate STRA8-positive spermatogonia. Bars = 50 μm. G) Graph depicts the average number of STRA8-positive spermatogonia in stages II to VI (Y axis) in either Cyp26a1/b1Flox/+;Cre control mice, Cyp26a1/b1Flox/Flox;Stra8−iCre mice, or Cyp26a1/b1Flox/Flox;Amh−Cre mice. *P < 0.05; **P < 0.01. Errors bars are SEM.

FIG. 6
FIG. 6

CYP26B1 activity is essential within the seminiferous epithelium for normal spermatogenesis. A–I) Representative cross-sections of testes from the Cyp26b1/dual Cre male mice 15 (A–C), 30 (D–F), and 90 (G–I) dpp and stained for STRA8 protein via immunohistochemistry (brown precipitate). The Flox/Flox;Cre-positive mutant line is shown in B, C, E, F, H, and I. Flox/+;Cre control animals are shown in A, D, and G. Representative cross-sections from testes collected from two different Flox/Flox;Cre-positive mutant animals are presented to show tubule-to-tubule and animal-to-animal phenotypic variations observed between mutant mice. Black arrows denote STRA8-positive preleptotene spermatocytes, and red arrows designate STRA8-positive spermatogonia; *degraded tubules; #tubules displaying spermatogenic recovery or unaffected tubules. Bars = 100 μm. J) Graph shows testis:body weight ratio (Y axis), measured in grams, for either Flox/+;Cre control animals (black bars) or Flox/Flox;Cre-positive conditional mutants (white bars) at either 15 or 90 dpp (X axis). Error bars are SEM. *P < 0.05. K–L) Images depict representative cross-sections of testes from Flox/Flox;Cre-positive Cyp26b1/dual Cre mutant male mice 15 (K) and 90 (L) dpp and stained for the germ-cell marker GCNA via immunohistochemistry (brown precipitate). Green arrows denote GCNA-positive germ cells and represent Sertoli cell-only tubules; #tubules show spermatogenic recovery or unaffected tubules. Bars = 100 μm.

Similar articles

Cited by

References

    1. Hogarth CA, Griswold MD. The key role of vitamin A in spermatogenesis. J Clin Invest. 2010;120:956–962. - PMC - PubMed
    1. McLean DJ, Russell LD, Griswold MD. Biological activity and enrichment of spermatogonial stem cells in vitamin A-deficient and hyperthermia-exposed testes from mice based on colonization following germ cell transplantation. Biol Reprod. 2002;66:1374–1379. - PubMed
    1. Mitranond V, Sobhon P, Tosukhowong P, Chindaduangrat W. Cytological changes in the testes of vitamin-A-deficient rats. I. Quantitation of germinal cells in the seminiferous tubules. Acta Anat (Basel) 1979;103:159–168. - PubMed
    1. Griswold MD, Bishop PD, Kim KH, Ping R, Siiteri JE, Morales C. Function of vitamin A in normal and synchronized seminiferous tubules. Ann N Y Acad Sci. 1989;564:154–172. - PubMed
    1. Van Pelt AM, De Rooij DG. The origin of the synchronization of the seminiferous epithelium in vitamin A-deficient rats after vitamin A replacement. Biol Reprod. 1990;42:677–682. - PubMed

Publication types

MeSH terms

Substances