Spring 2018 – Systems Biology of Reproduction Discussion Outline – Testis Systems Biology Michael K. Skinner – Biol 475/575 CUE 418, 10:35-11:50 am, Tuesday & Thursday February 22, 2018 Week 7 Testis Systems Biology Primary Papers: 1. Endo, et al. (2015) PNAS E2347-2356 2. Chalmel, et al., (2014) Plos One 9:e104418 3. Gatta, et al. (2010) BMC Genomics 11:401 Discussion Student 19: Reference 1 above • How do retinoids and Stra8 interact? • What experimental design was used? • What is the integration of retinoid actions and spermatogenesis? Student 20: Reference 2 above • What was the experimental design and omics technologies used? • What was the secretome observed? • How do Sertloli and germ cells interact? Student 22: Reference 3 above • What is the experimental and systems approach? • What genetic and expression relationships exist? • What insights are provided on the molecular control of male infertility? Periodic retinoic acid–STRA8 signaling intersects with PNAS PLUS periodic germ-cell competencies to regulate spermatogenesis Tsutomu Endoa,1,2, Katherine A. Romera,b,1, Ericka L. Andersona,c,1, Andrew E. Baltusa,c, Dirk G. de Rooija, and David C. Pagea,c,d,2 aWhitehead Institute, Cambridge, MA 02142; bComputational and Systems Biology Program and cDepartment of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139; and dHoward Hughes Medical Institute, Whitehead Institute, Cambridge, MA 02142 Contributed by David C. Page, March 24, 2015 (sent for review January 18, 2015; reviewed by William W. Wright) Mammalian spermatogenesis—the transformation of stem cells functionally pluripotent cells have been derived in vitro without into millions of haploid spermatozoa—is elaborately organized introduction of exogenous transcription factors or miRNAs (8). in time and space. We explored the underlying regulatory mecha- Undifferentiated spermatogonia periodically undergo spermato- nisms by genetically and chemically perturbing spermatogenesis in gonial differentiation (also known as the Aaligned-to-A1 transi- vivo, focusing on spermatogonial differentiation, which begins a tion) to become differentiating spermatogonia (also known as series of amplifying divisions, and meiotic initiation, which ends A1/A2/A3/A4/intermediate/B spermatogonia). During spermatogo- these divisions. We first found that, in mice lacking the retinoic acid nial differentiation, the spermatogonia down-regulate pluripotency- (RA) target gene Stimulated by retinoic acid gene 8 (Stra8), undif- associated genes (5, 9), lose capacity for self-renewal (4), and ac- ferentiated spermatogonia accumulated in unusually high numbers celerate their cell cycle (10) to begin a series of six transit-amplifying as early as 10 d after birth, whereas differentiating spermatogonia mitotic ivisions. At the conclusion of these mitotic divisions, were depleted. We thus conclude that Stra8, previously shown to be germ cells become spermatocytes, and undergo meiotic initiation required for meiotic initiation, also promotes (but is not strictly re- (Fig. 1A). This begins the meiotic program of DNA replication quired for) spermatogonial differentiation. Second, we found that and reductive cell divisions, ensuring that spermatozoa contribute injection of RA into wild-type adult males induced, independently, exactly one of each chromosome to the zygote. precocious spermatogonial differentiation and precocious meiotic Meiotic initiation is precisely coordinated with spermatogonial initiation; thus, RA acts instructively on germ cells at both transi- differentiation: The six mitotic divisions separating the two tions. Third, the competencies of germ cells to undergo spermato- gonial differentiation or meiotic initiation in response to RA were transitions occur over a span of exactly 8.6 d (11). Moreover, found to be distinct, periodic, and limited to particular seminiferous spermatogonial differentiation and meiotic initiation occur in stages. Competencies for both transitions begin while RA levels are close physical proximity. The testis comprises structures known low, so that the germ cells respond as soon as RA levels rise. To- as seminiferous tubules (Fig. S1A); while one generation of germ gether with other findings, our results demonstrate that periodic cells is initiating meiosis, a younger generation is simultaneously RA–STRA8 signaling intersects with periodic germ-cell competencies undergoing spermatogonial differentiation, within the same tubule to regulate two distinct, cell-type-specific responses: spermatogo- nial differentiation and meiotic initiation. This simple mechanism, Significance with one signal both starting and ending the amplifying divisions, contributes to the prodigious output of spermatozoa and to the As male sex cells mature into sperm, two pivotal transitions are elaborate organization of spermatogenesis. spermatogonial differentiation (exit from the stem cell pool) and meiotic initiation. These transitions occur in physical spermatogenesis | Stra8 | mouse | retinoic acid | testis proximity, with 8.6-d periodicity. We report that the gene Stra8, essential for meiotic initiation, also promotes (but is not he adult mammalian testis is among the body’s most pro- required for) spermatogonial differentiation. Moreover, in- Tliferative tissues, producing millions of highly specialized jected RA induces both transitions to occur precociously. We gametes, or spermatozoa, each day. Spermatogenesis (the pro- conclude that a periodic RA signal, acting instructively through gram of sperm production) is carefully regulated, ensuring that the common target Stra8, coordinates these transitions. This spermatozoa are produced at a constant rate. We used the mouse RA signal intersects with two distinct windows of sex-cell as a model to understand how mammalian spermatogenesis is competency, which both begin while RA levels are low; sex organized at the cellular and molecular level. We focused on cells respond quickly to rising RA. These mechanisms help ac- two key transitions: spermatogonial differentiation, which oc- count for the elaborate organization of sperm production, and curs cyclically and begins a series of programmed mitotic di- its prodigious output. visions, and meiotic initiation, which ends these divisions and marks the beginning of the meiotic program (Fig. 1A). Author contributions: T.E., K.A.R., E.L.A., D.G.d.R., and D.C.P. designed research; T.E., K.A.R., E.L.A., and A.E.B. performed research; T.E., K.A.R., E.L.A., and D.G.d.R. analyzed data; Like other proliferative tissues (e.g., blood, intestine, and and T.E., K.A.R., and D.C.P. wrote the paper. skin), the testis relies on a modest number of stem cells (1, 2). Reviewers included: W.W.W., Johns Hopkins Bloomberg School of Public Health. The undifferentiated spermatogonia (also known as the Asingle/ The authors declare no conflict of interest. Apaired/Aaligned spermatogonia), which encompass these stem Data deposition: The mRNA-sequencing dataset reported in this paper has been depos- cells, have a remarkable capacity for self-renewal and differen- ited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (acces- tiation: They can reconstitute spermatogenesis upon transplantation sion no. GSE67169). to a germ-cell-depleted testis (3, 4). In vivo, undifferentiated sper- 1T.E., K.A.R, and E.L.A. contributed equally to this work. matogonia ultimately give rise to a single cell type, spermatozoa, yet 2To whom correspondence may be addressed. Email: [email protected] or dcpage@wi. these undifferentiated spermatogonia express pluripotency-associ- mit.edu. BIOLOGY ated genes such as Lin28a (Lin-28 homolog A)(5)andPou5f/Oct4 This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. DEVELOPMENTAL (6, 7) and are the only postnatal mammalian cells from which 1073/pnas.1505683112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1505683112 PNAS | Published online April 20, 2015 | E2347–E2356 Both of these transitions require RA, a derivative of vitamin A A. In vitamin A-deficient (VAD) mice and rats, most germ cells Spermatids arrest as undifferentiated spermatogonia (16, 17). In VAD rat (steps 1-16) testes, some germ cells instead arrest just before meiosis, as preleptotene spermatocytes (16, 18). When VAD animals are Spermatocytes injected with RA or vitamin A, the arrested spermatogonia dif- (Pl, L, Z, P, D, SC2) ferentiate (17–19), and the arrested preleptotene spermatocytes Meiotic initiate meiosis (18). During spermatogonial differentiation, RA initiation Differentiating Cell differentiation is believed to act, at least in part, directly on germ cells: Sper- spermatogonia matogonia express RA receptors (RARs) (20), and genetic ab- (Adiff , In, B) lation of RARs in germ cells modestly impairs spermatogonial Spermatogonial Undifferentiated differentiation (21). differentiation spermatogonia To understand how RA might coregulate these two transi- (Aundiff ) tions, we needed to understand its target genes. During meiotic Mitotic cell division initiation, RA acts instructively, through the target gene Stra8 (Stimulated by retinoic acid gene 8). RA induces Stra8 expression B in germ cells—not in somatic cells—in both males and females Luminal (22, 23). Stra8 is required for meiotic initiation in both sexes: Stra8-deficient germ cells in postnatal males and fetal females arrest just before meiosis, without entering meiotic prophase (24, 1314 15 15 15 16 16 25). In contrast, no specific RA target genes have been impli- cated in spermatogonial differentiation: