Periodic Production of Retinoic Acid by Meiotic and Somatic Cells Coordinates Four Transitions in Mouse Spermatogenesis

Periodic production of retinoic acid by meiotic and somatic cells coordinates four transitions in mouse spermatogenesis Tsutomu Endoa,1,2, Elizaveta Freinkmana,3, Dirk G. de Rooija, and David C. Pagea,b,c,1 aWhitehead Institute, Cambridge, MA 02142; bDepartment of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139; and cHoward Hughes Medical Institute, Whitehead Institute, Cambridge, MA 02142 Contributed by David C. Page, October 4, 2017 (sent for review June 22, 2017; reviewed by Marvin L. Meistrich and Kyle E. Orwig) Mammalian spermatogenesis is an elaborately organized differenti- to become spermatozoa. Finally, these spermatozoa are released ation process, starting with diploid spermatogonia, which include into the lumen of seminiferous tubules. germ-line stem cells, and ending with haploid spermatozoa. The The four key transitions of spermatogenesis are precisely co- process involves four pivotal transitions occurring in physical prox- ordinated in time and space and occur in close physical and tem- imity: spermatogonial differentiation, meiotic initiation, initiation of poral proximity, cyclically, with an 8.6-d periodicity in mice (9). The spermatid elongation, and release of spermatozoa. We report how mouse testis is composed of structures known as seminiferous tu- the four transitions are coordinated in mice. Two premeiotic transi- bules (Fig. S1A); within tubule cross-sections, one sees stereotypical tions, spermatogonial differentiation and meiotic initiation, were collections or associations of germ cells at various steps of differ- known to be coregulated by an extrinsic signal, retinoic acid (RA). Our entiation (Fig. 1). The precise coordination of these steps is called the “cycle of the seminiferous epithelium” (or “seminiferous cy- chemical manipulations of RA levels in mouse testes now reveal cle”). In mice, researchers have characterized 12 distinct cellular that RA also regulates the two postmeiotic transitions: initiation of associations, known as seminiferous stages I to XII (10); the four spermatid elongation and spermatozoa release. We measured RA key transitions all occur in stages VII and VIII (10, 11) (Fig. 1 and concentrations and found that they changed periodically, as also Fig. S1 B–D). This intimate proximity of the four transitions to each reflected in the expression patterns of an RA-responsive gene, STRA8; other is largely conserved in other mammals, including humans RA levels were low before the four transitions, increased when the (12), rats (13, 14), hamsters (15), and rams (15). The layered gen- transitions occurred, and remained elevated thereafter. We found erations of germ cells in the seminiferous tubule are embedded in that pachytene spermatocytes, which express an RA-synthesizing and supported by somatic (Sertoli) cells that supply factors essential enzyme, Aldh1a2, contribute directly and significantly to RA produc- for spermatogenesis (16). We sought to investigate the cooccur- tion in testes. Indeed, chemical and genetic depletion of pachytene rence of the four key transitions, to understand the regulation of spermatocytes revealed that RA from pachytene spermatocytes was these transitions and the overall organization of spermatogenesis. required for the two postmeiotic transitions, but not for the two Both of the premeiotic transitions—spermatogonial differentia- premeiotic transitions. We conclude that the premeiotic transitions tion and meiotic initiation—require retinoic acid (RA), a derivative are coordinated by RA from Sertoli (somatic) cells. Once germ cells enter meiosis, pachytene spermatocytes produce RA to coordinate Significance the two postmeiotic transitions. In combination, these elements un- derpin the spatiotemporal coordination of spermatogenesis and en- Male mouse sex cells mature into sperm through a 35-d process sure its prodigious output in adult males. punctuated by four transitions, two occurring before meiosis (spermatogonial differentiation and meiotic initiation) and two retinoic acid | spermatogenesis | mouse | testis after meiosis (spermatid elongation and sperm release). The four transitions occur in proximity spatially and temporally, ammalian spermatogenesis is a choreographed process in with an 8.6-d periodicity. We describe how this coordination is Mwhich diploid spermatogonia undergo differentiation to achieved. The premeiotic transitions were known to be regu- give rise to specialized haploid gametes, called spermatozoa. lated by retinoic acid (RA). We show that RA also regulates the Within the testis, several key developmental transitions of two postmeiotic transitions. RA levels change periodically, and spermatogenesis occur in close physical and temporal proximity. meiotic cells contribute to its production. The two postmeiotic These transitions must be carefully regulated to ensure that large transitions require RA from meiotic cells while the premeiotic numbers of spermatozoa are produced continuously throughout transitions require RA from somatic cells. These elements un- reproductive life. We address the question of how these transi- derpin the spatiotemporal coordination of spermatogenesis to tions are coordinated at the cellular and molecular level. ensure constant sperm production throughout adult life. During spermatogenesis, four key transitions stand out: (i)differentiation of spermatogonia, (ii) meiotic initiation, (iii)initiation Author contributions: T.E., D.G.d.R., and D.C.P. designed research; T.E. and E.F. performed research; T.E., E.F., and D.G.d.R. analyzed data; and T.E. and D.C.P. wrote the paper. of spermatid elongation, and (iv) release of spermatozoa into the Reviewers: M.L.M., MD Anderson Cancer Center; and K.E.O., Magee-Womens lumen of seminiferous tubules (Fig. 1). In mice, spermatogenesis Research Institute. begins with undifferentiated type A spermatogonia, which include – Conflict of interest statement: K.E.O. and D.G.d.R. were coauthors on a 2016 paper; they the stem cells (1 4). Undifferentiated spermatogonia periodically worked on different aspects of the study and had no direct collaboration. undergo spermatogonial differentiation (also known as the Aaligned- This open access article is distributed under Creative Commons Attribution-NonCommercial- to-A1 transition) to become differentiating spermatogonia (also NoDerivatives License 4.0 (CC BY-NC-ND). known as A1/A2/A3/A4/intermediate/B spermatogonia). During 1To whom correspondence may be addressed. Email: [email protected] or spermatogonial differentiation, the spermatogonia lose the capac- [email protected]. ity for self-renewal (5) and begin a series of six transit-amplifying 2Present address: Immunology Frontier Research Center, Research Institute for Microbial mitotic divisions (6). Germ cells then become spermatocytes and Diseases, Osaka University, Suita 565-0871, Japan. undergo meiotic initiation (7, 8). DNA replication and two cell 3Present address: Metabolon, Inc., Research Triangle Park, NC 27709. divisions follow, resulting in the formation of haploid, round This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. spermatids, which elongate their nuclear and cytoplasmic contours 1073/pnas.1710837114/-/DCSupplemental. E10132–E10141 | PNAS | Published online November 6, 2017 www.pnas.org/cgi/doi/10.1073/pnas.1710837114 Downloaded by guest on September 30, 2021 PNAS PLUS Luminal RA concentration by absolute quantification, and we explored which cells provide the RA responsible for these four transitions by genetic and chemical cell depletion assays. We conclude that 13 14 15 15 15 16 1616 the two postmeiotic transitions are coordinated by RA produced by pachytene spermatocytes while the two premeiotic transitions are coordinated by RA produced by Sertoli cells. Results 1 2-3 4 5 6 7 8 9 10 11 12 RA Induces Release of Spermatozoa. We chemically manipulated RA levels in living testes to probe whether RA induces the release PPPPPPPPPDSC2 of spermatozoa. To ensure the efficacy of our experimental pro- tocols, we first confirmed that i.p. injection of exogenous RA raises the concentration of RA in testes, and, conversely, that injection of A In In / B B B / Pl Pl Pl / L L L / Z Z Z diff WIN18,446, an inhibitor of RA synthesis (30), lowers RA levels in testes. We monitored RA levels in testes both indirectly, by Adiff Adiff Adiff Adiff Adiff immunostaining for STRA8 as an RA-responsive marker (20, 31, 32), and directly, by measuring RA levels via liquid chromatog- A A A A A A A A A A A raphy/mass spectrometry (LC/MS). In control testis sections, undiff undiff undiff undiff undiff undiff undiff undiff undiff undiff undiff STRA8 protein was not detectable in germ cells in seminiferous I II-III IV V VI VII VIII IX X XI XII Basal stages V and VI (before key transitions) but was clearly present in Seminiferous stage spermatogonia and preleptotene spermatocytes in stages VII and 8.6 days VIII (during key transitions) (22) (Fig. S2A). As expected, at 1 d Release of spermatozoa after a single RA injection, STRA8 was strongly induced in stages V and VI. Conversely, after 2 d of daily WIN18,446 injections, Initiation of spermatid elongation STRA8 was not expressed in stages VII and VIII. LC/MS assays Initiation of meiosis confirmed that RA concentrations in testes were markedly in- Spermatogonial differentiation creased 1 d after RA injection and markedly decreased after 2 or 4 d of daily WIN18,446 injection (Fig. 2A). Fig. 1. Diagram of mouse spermatogenesis. Oakberg (10) identified 12 distinct

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