Retinol dehydrogenase 10 is indispensiblefor spermatogenesis in juvenile malesMing-Han Tong1, Qi-En Yang, Jeffrey C. Davis, and Michael D. Griswold1

School of Molecular Biosciences, Washington State University, Pullman, WA 99164

Edited by David C. Page, Whitehead Institute, Cambridge, MA, and approved November 26, 2012 (received for review August 29, 2012)

Retinoic acid (RA), an active vitamin A derivative, is essential formammalian spermatogenesis. Genetic studies have revealed thatoxidation of vitamin A to retinal by retinol dehydrogenase 10 (RDH10)is critical for embryonic RA biosynthesis. However, physiologicalroles of RDH10 in postnatal RA synthesis remain unclear, giventhat Rdh10 loss-of-function mutations lead to early embryonic le-thality. We conducted in vivo genetic studies of Rdh10 in postnatalmouse testes and found that an RDH10 deficiency in Sertoli cells,but not in germ cells, results in a mild germ cell depletion pheno-type. A deficiency of RDH10 in both Sertoli and germ cells in juve-nile mice results in a blockage of spermatogonial differentiation,similar to that seen in vitamin A-deficient animals. This defect inspermatogenesis arises from a complete deficiency in juvenile tes-ticular RA synthesis and can be rescued by retinoid administration.Thus, in juvenile mice, the primary, but not exclusive, source of RAin the testes is Sertoli cells. In contrast, adult Rdh10-deficient miceexhibit phenotypically normal spermatogenesis, indicating thatduring development a change occurs in either the cellular source ofRA or the retinaldehyde dehydrogenase involved in RA synthesis.

synchronous spermatogenesis | seminiferous epithelium | synchrony factor

Mammalian spermatogenesis begins shortly after birth, andcontinues to complete the first wave of spermatogenesis

until puberty. This provides the framework for subsequent sper-matogenic waves that occur in a continuous, well-coordinatedmanner in the adult (1–3). Spermatogenesis relies on the properfunction of spermatogonial stem cells, which are among the un-differentiated spermatogonia that include A single (As), A paired(Ap), and A aligned (Aal) spermatogonia (4). The undifferentiatedspermatogonia transform into A1 spermatogonia without a mitoticdivision. The A1 spermatogonia further generate, in succession,A2, A3, A4, In (intermediate), and B spermatogonia via a seriesof proliferative divisions. The differentiated spermatogonia thencontinue to differentiate into spermatocytes, haploid spermatids,and spermatozoa (4).Retinoic acid (RA), an active derivative of vitamin A [retinol

(Rol)], is essential for mammalian spermatogenesis (5–9). Vi-tamin A-deficient (VAD) rodents display a block at the transitionof the undifferentiated spermatogonia into A1 spermatogonia,resulting in seminiferous epithelium that contains only sper-matogonia and Sertoli cells. Administration of bioactive retinoidsto VAD animals reinitiates spermatogenesis in a synchronousmanner by releasing the block on spermatogonial differentiation(10–13). Recent studies have shown that Rol can induce sper-matogonial differentiation in cryptorchid testes as well (14). Nor-mally, testicular RA is synthesized in situ rather than originatingfrom the circulation (15, 16). To date, however, which mole-cules are involved in RA biosynthesis in the testes and whichcells are the primary sources of RA remain unknown.RA functions as a ligand for nuclear receptors [RA receptors

(RARs)], which bind to RA-response elements (RAREs) in reg-ulatory regions of target genes and control the expression of targetgenes (17). The RARs include three isotypes—Rara, Rarb, andRarg—that are widely expressed in the testes (18). RA is syn-thesized from an inactive precursor through a two-step enzymaticoxidation reaction in which Rol is first converted to retinaldehyde

(Ral) then to RA. The first step is mediated by two familiesof enzymes: the cytosolic alcohol dehydrogenase (ADH) family(e.g., ADH1, ADH3, ADH4), which are medium-chain dehy-drogenases/reductases, and Rol dehydrogenases (e.g., RDH1,RDH10, RDH11, RDH13, RDH14, RDH15), which are short-chain dehydrogenases/reductases (19, 20). Enzymes belongingto the Ral dehydrogenase (RALDH) family are responsible forthe second step of RA synthesis. The RALDH family includesRALDH1, RALDH2, and RALDH3, which are encoded byAldh1a1, Aldh1a2, and Aldh1a3, respectively (21, 22). Studieswith gene loss-of-function and RA reporter mice have suggestedthat RDH10 and RALDH2 are critical for RA biosynthesis inthe embryo (22–26). In contrast to its well-established importance inthe embryo, the physiological roles of RDH10 in postnatal RAsynthesis remain elusive, given that Rdh10 loss of function resultsin early embryonic lethality.To address this question, we generated testicular cell-specific

conditional knockout (cKO) alleles of Rdh10, which permittedan evaluation of RDH10 on postnatal testicular RA synthesis.We report here that Rdh10 loss of function in both Sertoli andgerm cells or only in Sertoli cells leads to a defect in spermato-gonial differentiation in juvenile mice. Through RA reportermouse analyses, we found that Rdh10 loss of function in bothSertoli and germ cells completely impairs testicular RA sig-naling in juvenile animals. Interestingly, spermatogenesis wasprogressively recovered in adult Rdh10 cKO mice. These findingsdemonstrate that RDH10 is essential for juvenile spermatogenesis,but not for adult spermatogenesis.

ResultsMale Rdh10 Mutants Display Impaired Spermatogenesis. To determinethe role of Rdh10 in postnatal mouse development and disease,we used a cKO strategy that generated an Rdh10 floxed line(Rdh10fl/fl), in which exon 2 of the Rdh10 allele is flanked by loxPsites (Fig. S1). Global inactivation of Rdh10 causes embryoniclethality (24–26). Affymetrix array data and in situ hybridizationfindings revealed Rdh10 expression in postnatal mouse testes, in-cluding Sertoli cells and germ cells (Tables S1 and S2 and Fig. S2).To explore the role of RDH10 in postnatal testes, we deletedRdh10 in Sertoli cells using the Amh-Cre transgenic line (Rdh10fl/fl,Amh-Cre+) (27), in germ cells using the Stra8-Cre transgenicline (Rdh10fl/fl, Stra8-Cre+) (28), or in both Sertoli and germcells (Rdh10fl/fl, Amh-Cre+, Stra8-Cre+). Amh-Cre transgenic miceexhibit high recombinase activity as early as embryonic day 14.5(27). Stra8-Cre male mice start to express Cre in spermatogoniaat postnatal day 3 (3 dpp), whereas female germ cells do not ex-press Cre (28). To confirm the Cre expression in mutants, we

Author contributions: M.-H.T. and M.D.G. designed research; M.-H.T., Q.-E.Y., and J.C.D.performed research; M.-H.T. contributed new reagents/analytic tools; M.-H.T., Q.-E.Y.,J.C.D., and M.D.G. analyzed data; and M.-H.T. and M.D.G. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence may be addressed. E-mail: minghan@wsu.edu or mgriswold@wsu.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1214883110/-/DCSupplemental.

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generated compound mutant mice by crossing the Cre micewith a double-fluorescent reporter line, ROSA26mTmG (29).Membrane-targeted EGFP allowed visualization of cellular pat-terns of testicular Cre activity, demonstrating Sertoli cell- or germcell-specific Cre expression, consistent with previous reports (27,28). To avoid global deletion of the Rdh10 floxed allele, weused only Rdh10fl, Stra8-Cre+ females for breeding. For thisstudy, we designated the four different genotypes of mice ascontrol (Rdh10fl/fl), Rdh10 gKO (germ cell-specific KO;Rdh10fl/fl, Stra8-Cre+), Rdh10 sKO (Sertoli cell-specific KO;Rdh10fl/fl, Amh-Cre+), and Rdh10 sgKO (Sertoli and germ cell-specific KO; Rdh10fl/fl, Amh-Cre+, Stra8-Cre+).To explore the role of Rdh10 in the testes of juvenile males, we

collected testes from mice at age 2-3 wk. There were no signif-icant differences in body weight among the four genotypes. Therelative weights of testes were significantly lower in Rdh10 sKOand sgKO mice compared with controls at both 2 wk and 3 wk [atage 2 wk: control, 0.243 ± 0.021 (mean ± SD testes weight/bodyweight × 100); Rdh10 sKO, 0.170 ± 0.003, P < 0.05; Rdh10sgKO, 0.124 ± 0.009, P < 0.01; at age 3 wk: control, 0.249 ±0.028; Rdh10 sKO, 0.180 ± 0.035, P < 0.05; Rdh10 sgKO, 0.133 ±0.011, P < 0.01]. In contrast, there were no significant differencesbetween controls and Rdh10 gKO (at age 2 wk: Rdh10 gKO,0.246 ± 0.010, P = 0.51; at age 3 wk: Rdh10 gKO, 0.250 ± 0.007,P = 0.49) (Fig. 1 A and B). In addition, the testes weight indexwas dramtically lower at both 2 and 3 wk in Rdh10 sgKO mutantscompared with Rdh10 sKO mutants (P < 0.05). Histologicalanalyses showed that the control testes (Fig. 1 C and G) andRdh10 gKO mutant testes (Fig. 1 E and I) were indistinguish-able; at 2 wk, both genotypes had spermatogonia and preleptene,leptene, zygotene, and pachytene spermatocytes, and by 3 wk,round spermatids had formed in both genotypes. In contrast,in the Rdh10 sgKO testes, most seminiferous tubules (>85%)were devoid of meiotic cells and retained only undifferentiatedspermatogonia-like cells and Sertoli cells (Fig. 1 D and H). Thesedefects were first apparent in the 7-d-old juveniles. An Rdh10deficiency exclusively in Sertoli cells (Rdh10 sKO) had milderphenotypes with ∼45% degenerated tubules (Fig. 1 F and J).Numerous degenerated germ cells were present in the Rdh10

sgKO and sKO testes (Fig. S3 A–D). A TUNEL assay detectedmore TUNEL-positive germ cells in Rdh10 sKO and sgKOmutant testes than in control and gKO testes (Fig. S3 E–H).

Rdh10 Deficiency in both Sertoli and Germ Cells Causes Blockage ofDifferentiation of Aal Spermatogonia into A1 Spermatogonia. Theremaining germ cells in Rdh10 sgKO testes were immunostainedwith the molecular markers of differentiated spermatogonia andspermatocytes. A marker for the formation of DNA double-strand breaks in early meiosis, γ-H2AX (30), was absent withinmost seminiferous tubules of Rdh10 sgKO testes of 2-wk-old(Fig. 2 D and F) and 3-wk-old mutants (Fig. S4 D and F) but waspresent in control testes (Fig. 2 A and C and Fig. S4 A and C),suggesting noninitiation of meiosis in the germ cells of Rdh10sgKO testes. Immunostaining for STRA8, a marker for differen-tiated spermatogonia (also a RA-responsive gene), was done todetermine whether spermatogonia in Rdh10 sgKO testes differ-entiate. As expected, control testes contained many seminiferoustubules with STRA8-positive germ cells (Fig. 2 G and I), butSTRA8-positive cells were rarely observed in Rdh10 sgKO testes

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Fig. 1. Impaired spermatogenesis occurs in Rdh10 sgKO and sKO juvenilemice. (A) Gross morphology of representative testes from a 2-wk-old controland age-matched Rdh10 gKO, sKO, and sgKO mutants. (B) Comparisons oftestis weight from 2- or 3-wk-old controls and mutants (n = 4–8 for eachgenotype per data point). (C–J) H&E staining of control (C and G), Rdh10sgKO (D and H), gKO (E and I), and sgKO (F and J) testes at age 2 or 3 wk.(Scale bars: 20 μm.)

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Fig. 2. Decreased expression of markers for spermatogonial differentiationin Rdh10 sgKO mutant testes. (A–F) Immunofluorescence staining for γH2AX(red) in sections of 2-wk-old control (A–C) and Rdh10 sgKO (D–F) testes, withcostaining for germ cell marker GCNA (green). (G–L) Immunohistochemicalstaining for STRA8 (red) in sections of 2-wk-old control (G–I) and Rdh10 sgKO(J–L) testes, with costaining for germ cell marker GCNA (green). (M) Quanti-tative RT-PCR analysis of mRNA levels of markers for spermatogonial differ-entiation in control, Rdh10 sgKO, sKO, and gKO mutant testes at age 2 wk.Data are expressed as mean ± SD fold differences compared with controls,normalized to Rps2. n = 4–6; Student t test. (Scale bars: 20 μm.)

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from 2-wk-old (Fig. 2 J and L) and 3-wk-old mutants (Fig. S4 Jand L), indicating nondifferentiation of spermatogonia. Weperformed concomitant immunostaining for GCNA, a germ cell-specific maker (31), to differentiate between somatic cells andgerm cells in the testes. Consistent with the foregoing histologicalstudy, the GCNA staining showed that only one layer of germ cellswas located along the basement membrane in Rdh10 sgKO testes(Fig. 2 E and K and Fig. S4 E and K), in contrast to the multiplelayers of germ cells within the seminiferous epithelium of controltestes (Fig. 2 B and H and Fig. S4 B and H). Control testes con-tained seminiferous tubules with KIT-positive germ cells, whereasKIT-positive cells were rarely observed in Rdh10 sgKO testesfrom 2-wk-old mice (Fig. S5). Collectively, the observed defectsin Rdh10 sgKO testes phenocopy the abnormalities observed inVAD animals.We used levels of mRNA for key markers for spermatogonial

differentiation and meiotic initiation to examine the differentiationof spermatogonia in Rdh10 sgKO testes. Markers of differentiated(A1) spermatogonia included Stra8 and c-kit. Markers of earlymeiotic prophase included Spo11, which encodes a topoisomerase(32); Dmc1, which encodes a meiosis-specific recombinase (33);and Sycp3, which encodes a component of the synaptonemalcomplex (34). mRNA levels of spermatogonial differentiationmarkers (Stra8 and c-kit) and meiosis markers (Spo11, Dmc1,and Sycp3) were significantly reduced in Rdh10 sgKO mutanttestes and even in Rdh10 sKO testes, as measured by quantitativeRT-PCR (Fig. 2M). Taken together, these data clearly indicate thatspermatogonia in Rdh10 sgKO testes do not enter differentiationand thus do not initiate meiosis.To further characterize the state of the remaining spermato-

gonia, we used staining with an antibody to an undifferentiatedspermatogonial marker, PLZF. The number of PLZF-positivespermatogonia was significantly elevated in Rdh10 sgKO testes (Fig.3 A–C). We then performed immunostaining for SOX9, a markerfor Sertoli cells (35), to determine the state of Sertoli cells in Rdh10sgKO. The numbers of Sertoli cells in control and Rdh10 sgKOtestes did not differ significantly [control, 32.58 ± 1.94 (mean ±SD SOX9+/tubule); sgKO, 33.05 ± 1.95; n= 3; P > 0.05] (Fig. S6).We administered a short-duration (2 h) 5-ethynyl-2′-deoxyuridine(EdU) pulse to control and Rdh10 sgKO mutant mice, and founda lower ratio of both EdU-positive and PLZF-positive cells toPLZF-positive cells (EdU+PLZF+/PLZF+) in Rdh10 sgKO

testes compared with controls (Fig. 3 D–F), indicating lower pro-liferation rates in these PLZF-positive spermatogonia. A TUNELassay on control and Rdh10 sgKO testes revealed little if anydifference in apoptosis of PLZF-positive spermatogonia [control,1.6 ± 0.51 (mean ± SD TUNEL+PLZF+/PLZF+ × 100); sgKO,1.9 ± 0.46; n = 3–5; P = 0.21]. Taken together, these findingsprovide strong evidence that the accumulation of undifferentiated(PLZF-positive) spermatogonia in the Rdh10 sgKO testes resultsfrom blockage of the differentiation of Aal into A1 spermatogonia,rather than from hyperproliferation and/or decreased apoptosis.

RDH10 Deficiency Affects RA Signaling in Testis.RDH10 is known tobe essential for the synthesis of embryonic Ral, the intermediatemetabolite in RA biosynthesis (24–26). We used a RARElacZreporter line harboring a RA-responsive transgene, which allowsvisualization of the distribution of RA signaling by X-Gal staining,to examine RA levels in the testes of the mutant mice (36). Wefound no lacZ activity in testes from Rdh10 sgKO mutants, butstrong lacZ staining in the control testes (Fig. 4A). We thenverified impaired RA signaling in the Rdh10 sgKO mutant testesby treating 2-wk-old Rdh10 sgKO mice with RA for 24 h. TheRdh10 sgKO testes demonstrated rescue of lacZ expression after

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Fig. 3. Accumulation of PLZF-positive spermatogonia in Rdh10 sgKO mu-tant testes. (A and B) Immunofluorescence staining for PLZF (red) in sectionsof 2-wk-old control (A) and Rdh10 sgKO (B) testes. (Scale bar: 20 μm.) (C)Quantification of PLZF-positive cells per seminiferous tubule in 2-wk-oldtestes. (D and E) Immunostaining for PLZF (green) and EdU (red) in sectionsof 2-wk-old control (D) and Rdh10 sgKO (E) testes. Arrowheads indicate bothPLZF- and EdU-positive spermatogonia (orange). (Scale bar: 20 μm.) (F )Quantification of proliferative spermatogonia in control and Rdh10 sgKOtestes at age 2 wk. The number of EdU-positive cells per number of PLZF-positive cells was recorded. All seminiferous tubules at each section werecounted (n = 3–5). Error bars represent SD. *P < 0.05, Student t test.

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Fig. 4. Expression of RA-responsive genes in Rdh10 sgKO testes. (A) X-Galstaining of testes and kidneys from Rdh10 sgKO, RA-rescued Rdh10 sgKO,and control littermates harboring a RARE-Hspa1b-lacZ transgene at age 2 wk.X-Gal staining of kidneys from mutants and controls served as a positivecontrol, and X-Gal staining of WT without a RARE-Hspa1b-lacZ transgeneserved as a negative control. (B) Histological analysis of X-Gal staining oftestes from Rdh10 sgKO, RA-rescued Rdh10 sgKO, and control littermatesharboring a RARE-Hspa1b-lacZ transgene at age 2 wk. Cells with a positivesignal are shown in blue. Nuclei were counterstained with fast red. (Scalebar: 20 μm.) (C and D) Quantitative RT-PCR analysis of mRNA levels ofRA-induced genes (C ) and RA-repressed genes (D) in Ral-rescued Rdh10sgKO testes at age 2 wk. Data are expressed as mean ± SD fold differencescompared with controls (Rdh10 sgKO), normalized to Rps2. n = 3. *P < 0.05,Student t test.

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RA treatment (Fig. 4A), indicating that these testes lacked RA.Histologically, seminiferous tubes in control testes containedlacZ-positive spermatogonia and spermatocytes (Fig. 4B and Fig.S7A). Significantly, virtually no lacZ-positive cells were found inRdh10 sgKO testes (Fig. 4B and Fig. S7B), but >80% of theseminiferous tubes in these testes contained lacZ-positive sper-matogonia after exposure to RA (Fig. 4B and Fig. S7C).We treated RDH10 sgKO mice with Ral in an attempt to rescue

the phenotype. Exogenous Ral administration significantly in-duced expression of Stra8 and Kit in Rdh10 sgKO mutant testes,as determined by quantitative RT-PCR (Fig. 4C). In addition,expression of Lin28 and Mycn, which are RA-repressed genes(37, 38), was dramatically inhibited after RA treatment (Fig. 4D).Taken together, these data support the idea that testicular cell-specific Rdh10 deficiency impairs RA signaling, and thus Rdh10sgKO spermatogenesis defects result from the lack of an RA signal.

Retinoid Induces Synchrony of Spermatogenesis in Rdh10 sgKOMutants. Spermatogenesis in mammals is highly coordinated pro-cess of germ cell differentiation occurring in the seminiferoustubules, where various types of germ cells form well-defined cel-lular associations (39). These typical cell associations are stages ofthe cycle of the seminiferous epithelium, 12 of which are presentin the mouse (39). VAD male mice exhibit arrested spermatogo-nial differentiation; however, administration of vitamin A or RAto VAD animals results in the reinitiation of spermatogenesis,such that the epithelium becomes stage-synchronized. Given thata loss of RDH10 in mouse testes causes testicular defects similarto those seen in VAD mice, we examined whether RA or Raltreatment also induces synchronization of the seminiferous epi-thelium in Rdh10 sgKO mutant mice.In this experiment, 3-wk-old control and Rdh10 sgKO mutant

male mice received a single RA or Ral injection and were thenfed a normal vitamin A-containing diet for 35 d. Most semi-niferous tubules from representative RA-treated Rdh10 sgKOmales were enriched in stages VII–IX of the cycle of the semi-niferous epithelium, whereas retinoid treatment did not enrichthe stages of the seminiferous epithelial cycle in any controlindividuals (Fig. 5 A and B). STRA8 immunostaining furtherdemonstrated the enrichment of stage VIII in the RA-treatedRdh10 sgKO mutants (Fig. 5 C and D). Stage frequency analysisand synchrony factor calculations confirmed that spermatogen-esis was more synchronous in RA-treated mutants than in RA-treated controls (Fig. 5 E and F). Results after Ral treatmentwere similar to those after RA treatment, whereas Rol treat-ment cannot induce synchronized spermatogenesis in mutants(mean synchrony factor, 1.08 ± 0.09; n = 3; P > 0.05).

Spermatogenesis Is Recovered in Adult Rdh10 sgKO Mutant Males.Given the importance of Rdh10 in regulating juvenile spermato-genesis, we next investigated the requirement for Rdh10 in sup-port of adult spermatogenesis. We found that Rdh10 sgKO malesaged <7 wk were infertile or subfertile, but those aged >9 wkwere fertile. Moreover, litters produced from controls and mutantsdid not differ in size. Histologically, spermatogenesis in mostseminiferous tubules from Rdh10 sgKO mutants was reinitiated atage 4 wk, because spermatocytes were present (Fig. 6). Spermatidswere rarely observed in 5- and 6-wk-old Rdh10 sgKO testes (Fig. 6).By age 9 wk, spermatogenesis was completed in Rdh10 sgKOtestes, with all seminiferous tubules containing the expected sper-matogonia, spermatocytes, and spermatids (Fig. 6). Examination ofrecovery of spermatogenesis in adult Rdh10 sgKO males showedthat only 1 out of 48 exhibited spermatogenesis with an alteredstage frequency.

DiscussionIn this study, we investigated the biological function of Rdh10in postnatal mouse testes using a conditional targeted deletion,

and found that Rdh10 is required for spermatogenesis in juve-niles. Specifically, we found that Rdh10 is essential for juveniletesticular RA biosynthesis (i.e., generation of Ral for use as asubstrate for RALDH synthesis of RA), and thus that the testiculardefects in Rdh10 mutants result from lack of RA. Interestingly,we found that Rdh10 in Sertoli and/or germ cells is dispensablefor spermatogenesis in the adult.In mammals, under physiological conditions, testicular RA

derives from local synthesis within the seminiferous tubule ratherthan from the circulation, because of the presence of a metabolicbarrier (15, 18). Testicular RA is synthesized in a two-step pro-cess from Rol provided either by the circulation or by the localstores of retinyl esters. The initial step, conversion of Rol intoRal, is the rate-limiting and reversible step in RA synthesis (40).We found that Rdh10 is expressed in both mouse Sertoli cellsand spermatogonia. This finding, together with a wealth of evi-dence that RDH10-mediated Ral production is a critical step inembryonic RA synthesis, leads us to postulate that RDH10 maybe responsible for the postnatal testicular RA synthesis. The datapresented here support this hypothesis in several respects. First,loss of the RDH10 enzyme either in both Sertoli cells and germcells or only in Sertoli cells results in defects in spermatogonialdifferentiation characterized by the presence of only Sertoli cellsand undifferentiated spermatogonia within the seminiferoustubules. This phenotype is identical to the testicular abnormali-ties observed in VAD mice. Second, a lack of RDH10 activity inRARElacZ reporter mice causes complete loss of lacZ activityin testes; however, RA administration rescues expression of thereporter in mutant testes. Finally, retinoid supplementation notonly reinitiates spermatogonial differentiation, but also induces

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Fig. 5. Synchrony of spermatogenesis in Rdh10 sgKO testes after RA treat-ment. (A and B) Hematoxylin staining of representative testes from 3-wk-oldcontrol (A) and Rdh10 sgKO (B) mice with a vitamin A-containing diet for5 wk, after an RA injection. Roman numerals indicate the stage of theseminiferous epithelial cycle. (C and D) Immunostaining for STRA8 (red) insections of representative testes from 3-wk-old control (C) and Rdh10 sgKO(D) mice after RA treatment. The seminiferous epithelium contains STRA8-positive cells, a characteristic marker of stage VIII. (E ) Stage frequency ofseminiferous epithelial cycle in control and Rdh10 sgKO individuals afterRA treatment. (F ) Calculated synchronization factors of control and Rdh10sgKO mice after RA treatment. n = 5–8. Error bars represent SD. *P < 0.05,Student t test. (Scale bars: 100 μm.)

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synchrony of spermatogenesis in Rdh10 sgKO mutants, similarto what occurs in RA-rescued VAD rodents. Thus, although manyenzymes displaying all-trans- and/or cis-Rol dehydrogenase, such asRDH11, RDH13, RDH14, ADH1, ADH3, and ADH4 (41), areexpressed in juvenile mouse testes, it is clear that the RDH10enzyme contributes significantly to physiological synthesis of RAduring juvenile spermatogenesis.It is interesting to note that Sertoli cell-specific Rdh10 de-

ficiency could result in defects in spermatogonial differentiation,although the phenotype is not as severe as that seen with a de-ficiency in both Sertoli cells and germ cells. In contrast, germcell-specific Rdh10-deficient juvenile mice demonstrated normalspermatogenesis. These findings suggest that both Sertoli cellsand germ cells produce Ral by RDH10, but Sertoli cells may bethe major source of Ral in juvenile mouse testes. Ral is sub-sequently converted into RA in Sertoli cells, which express Aldh1a1and Aldh1a2 in juvenile mouse testes (18). The fact that Rdh10sgKO mutants can be rescued by Ral treatment, which exposesall cells of the body to Ral, suggests that spatially restricted avail-ability of Ral is not critical for spermatogenesis. This suggestionis consistent with the observation that embryo development isnot dependent on localized Ral production (24, 25).A notable finding is that spermatogenesis in Rdh10 sgKO

mutants was reinitiated starting at age 4 wk and was completedby age 9 wk. In one study, Ral-rescued Rdh10 global KO micesurvived to adulthood and were fertile (24). Although the status ofspermatogenesis was not reported in that study, it is possible—aswe have demonstrated in Rdh10 sgKO mice—that spermato-genesis was also impaired in the juvenile Rdh10 global KO miceand then recovered in adult mice. The ability of the Rdh10 sgKOadult mice to recover from early impaired spermatogenesis sug-gests that other Rol-oxidizing enzymes must be involved. Forexample, ADH KO studies have demonstrated a postnatal rolefor ADHs in Rol metabolism, although in these KOs, even Adh-del compound KOs lacking all six ADHs (Adh1, 2, 3, 4, 5a, and5b), were fertile (42). ADHs and/or other RDHs are ex-pressed along with RDH10 in the testes. In the absence of RDH10,they could be the source of Ral in the testes. In addition,β-carotene 15,15′-monoxygenase, which can produce Ral fromprovitamin A, is expressed in >20-d-old mouse testes, suggestingthat it could play a role in the recovery seen in Rdh10 sgKO adulttestes (18, 43). Alternatively, the testes could obtain Ral for RAsynthesis from the circulation. Nonetheless, it is clear that theseenzymes do not compensate for the absence of RDH10-mediatedRal production in juvenile mouse testes.Finally, considering the defects in spermatogenesis in juvenile

mice that were recovered in adults, our Rdh10-deficient mousemodel is a unique model that can provide insight into the RAsignaling pathways that control spermatogonial differentiationand meiotic initiation and regulate formation of the seminiferousepithelial cycle.

Materials and MethodsGeneration of Rdh10 cKO Mice. Rdh10-targeted ES cells were generated bythe University of California Davis Knockout Mouse Project Repository. Thetargeting allele was designed for conditional mutagenesis using the FLP-FRTand Cre-loxP system (Fig. S1). A splice acceptor lacZ gene trap cassette flankedby an FRT site was inserted between exon 1 and exon 2 of the Rdh10 gene.The loxP sites were introduced to flank exon 2 of the Rdh10 gene. Excision ofexon 2 creates a frameshift mutation in RDH10. We used a correctly targetedES cell clone for injection into C57BL6/J (B6) blastocysts. Resulting chimericmale mice were mated to WT B6 mice, and progeny were screened by PCRfor germ line transmission of the targeted allele. Mice were then bred withACTB-FLP transgenic mice (44) to delete the gene trap cassette and obtainRdh10 floxed mutants. The homozygous Rdh10 floxed mutants (Rdh10fl/fl)were viable and fertile. Rdh10 floxed mutants were then crossed with cell-specific expressed Cre mice for excising the loxP-flanked exon 2 to generatecell specific Rdh10 KO mice. All animal experiments were performed in accor-dance with the National Institutes of Health’s Guide for the Care and Use ofLaboratory Animals, and all protocols were approved by Washington StateUniversity’s Animal Care and Use Committee.

Histological, Immunohistochemical, and TUNEL Analyses. Testes were fixed inBouin’s solution, embedded in paraffin, and sectioned. Sections were depar-affinized, rehydrated, and stained with H&E. For immunohistochemical stud-ies, slides were boiled in 10 mM sodium citrate buffer (pH 6.0) for 15 min,brought to room temperature, washed in PBS with 0.1% Triton X-100, andthen incubated for 60 min at room temperature with blocking buffer (10%donkey serum, 1% BSA, and 0.1% Triton X-100 in PBS). The sections werethen incubated with a 1:50 dilution of rat anti-GCNA IgM (kindly providedby Dr. G. Enders, University of Kansas, Kansas City, KS) and a 1:200 dilutionof mouse anti-γH2AX IgG (Millipore), rabbit anti-STRA8 IgG, rabbit anti-PLZF(Santa Cruz Biotechnology), or rabbit anti-SOX9 IgG (Millipore), or a 1:50dilution of rat anti-KIT (Millipore) overnight at 4 °C. The slides were thenwashed with PBS, and Alexa Fluor 488- and Alexa Fluor 594-conjugateddonkey secondary antibody (Jackson ImmunoResearch Laboratories) wereadded at a 1:500 dilution. After 60 min at room temperature, the sectionswere washed in PBS, rinsed quickly in pure ethanol, mounted in ProlongGold Antifade medium with DAPI (Molecular Probes), and then analyzedby fluorescence microscopy (Olympus). Apoptotic cells were detected usingan In Situ Cell Death Detection Kit, Fluorescein (Roche Applied Science)according to the manufacturer’s instructions.

Retinoid Administration and Stage Analyses. For short-term treatment, 2-wk-old animals were injected i.p. with all-trans RA, all-trans Ral, or all-trans Rol(Sigma-Aldrich; 400 μg per injection) with vehicle as controls twice in a 24-h period, and testes were collected at 24 h posttreatment for either RNAextraction or histological studies. For resuming spermatogenesis, 3-wk-old mice received one 500-μg i.p. injection of all-trans-RA, all-trans-Rol, orall-trans-Ral (Sigma-Aldrich) and were then fed a a normal diet until beingkilled. Testes were fixed in Bouin’s solution, embedded in paraffin, and sec-tioned at 5 μm. Sections were then stained with hematoxylin to identify thestages of the seminiferous epithelium according to previously establishedcriteria (45). Stage frequency and synchronization factor were then de-termined using the methods described by Bianchi and Tiglao (46) and Siiteriet al. (47), respectively.

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Fig. 6. Histological analysis of testes from peripubertal and adult control (A–D) and Rdh10 sgKO (E–H) mice. (Scale bars: 20 μm.)

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EdU Labeling. Juvenile mice were injected i.p. with EdU (1 mg per injection;Invitrogen) in PBS. The mice were euthanized 2 h later, and testes werefixed in 4% paraformaldehyde/PBS solution, embedded in paraffin, andsectioned. After the sections were immunostained with PLZF or γH2AX,EdU incorporation was detected using the Click-It EdU Alexa Fluor 594Imaging Kit (Invitrogen) according to the manufacturer’s protocol.

Quantitative RT-PCR Assays. Total RNA was extracted using TRIzol reagent(Invitrogen) and treated with DNaseI (Ambion). Total RNA was reverse-transcribed using an iScript cDNA Synthesis Kit (Bio-Rad). Quantitative RT-PCRwas performed using Fast SYBR Green PCR Master Mix (Applied Biosystems)on a 7500 Fast PCR System (Applied Biosystems). Relative gene expression wasanalyzed by the comparative CT method, using ribosomal protein S2 (Rps2) asa normalized control. RT-PCR primer sequences were described in Table S3.

X-Gal Staining. Rdh10fl/fl, Amh-Cre+ (male) and Rdh10fl/fl, Stra8-Cre+ (female)mice were crossed with RARElacZ reporter line harboring RARE-Hspa1b-lacZ

alleles to obtain Rdh10fl/+, Amh-Cre+, RARElacZ; Rdh10fl/+, Stra8-Cre+, RARElacZ;and Rdh10fl/+, RARElacZ mice (36). The Rdh10fl/+, Amh-Cre+, RARElacZ;Rdh10fl/+, Stra8-Cre+, RARElacZ (female); and Rdh10fl/+, RARElacZ mice wereused to produce Rdh10fl/fl, Amh-Cre+, RARElacZ; Rdh10fl/fl, Stra8-Cre+, RARElacZ;and Rdh10fl/fl, RARElacZ mice. The Rdh10fl/fl, Amh-Cre+, RARElacZ maleswere bred to Rdh10fl/fl, Stra8-Cre+, RARElacZ females to generate Rdh10fl/fl,Stra8-Cre+, Amh-Cre+, RARElacZ and Rdh10fl/fl, RARElacZ mice. Testes, epididy-mides, and kidneys from mice bearing RARE-Hspa1b-lacZ alleles were fixed in4% paraformaldehyde in PBS for 2 h at room temperature, washed, stainedwith X-Gal at 37 °C overnight, washed, and then photographed. The stainedtestes were then processed, embedded in paraffin, and sectioned. Sectionswere counterstained with fast red.

ACKNOWLEDGMENTS. We thank Dr. G. Enders for the anti-GCNA antibodyand Ms. Elizabeth B. Evans for critically reading the manuscript. This workwas supported by National Institutes of Health Grants HD10808 (to M.D.G.)and HD06777 (to M.-H.T. and M.D.G.).

1. Kluin PM, Kramer MF, de Rooij DG (1982) Spermatogenesis in the immature mouseproceeds faster than in the adult. Int J Androl 5(3):282–294.

2. Bellvé AR, et al. (1977) Spermatogenic cells of the prepuberal mouse: Isolation andmorphological characterization. J Cell Biol 74(1):68–85.

3. Yoshida S, et al. (2006) The first round of mouse spermatogenesis is a distinctiveprogram that lacks the self-renewing spermatogonia stage. Development 133(8):1495–1505.

4. de Rooij DG (2001) Proliferation and differentiation of spermatogonial stem cells.Reproduction 121(3):347–354.

5. Wolgemuth DJ, Chung SS (2007) Retinoid signaling during spermatogenesis as re-vealed by genetic and metabolic manipulations of retinoic acid receptor alpha. SocReprod Fertil Suppl 63:11–23.

6. Griswold MD, et al. (1989) Function of vitamin A in normal and synchronized semi-niferous tubules. Ann N Y Acad Sci 564:154–172.

7. Clagett-Dame M, Knutson D (2011) Vitamin A in reproduction and development.Nutrients 3(4):385–428.

8. Wilson JG, Roth CB, Warkany J (1953) An analysis of the syndrome of malformationsinduced by maternal vitamin A deficiency: Effects of restoration of vitamin A atvarious times during gestation. Am J Anat 92(2):189–217.

9. Wolbach SB, Howe PR (1925) Tissue changes following deprivation of fat-soluble Avitamin. J Exp Med 42(6):753–777.

10. Huang HF, Hembree WC (1979) Spermatogenic response to vitamin A in vitamin A-deficient rats. Biol Reprod 21(4):891–904.

11. van Pelt AM, de Rooij DG (1990) Synchronization of the seminiferous epithelium aftervitamin A replacement in vitamin A-deficient mice. Biol Reprod 43(3):363–367.

12. Van Pelt AM, De Rooij DG (1990) The origin of the synchronization of the seminif-erous epithelium in vitamin A-deficient rats after vitamin A replacement. Biol Reprod42(4):677–682.

13. Morales C, Griswold MD (1987) Retinol-induced stage synchronization in seminiferoustubules of the rat. Endocrinology 121(1):432–434.

14. Sugimoto R, Nabeshima Y, Yoshida S (2012) Retinoic acid metabolism links theperiodical differentiation of germ cells with the cycle of Sertoli cells in mouseseminiferous epithelium. Mech Dev 128(11-12):610–624.

15. Kurlandsky SB, Gamble MV, Ramakrishnan R, Blaner WS (1995) Plasma delivery ofretinoic acid to tissues in the rat. J Biol Chem 270(30):17850–17857.

16. Kane MA, Folias AE, Wang C, Napoli JL (2008) Quantitative profiling of endogenousretinoic acid in vivo and in vitro by tandem mass spectrometry. Anal Chem 80(5):1702–1708.

17. Bastien J, Rochette-Egly C (2004) Nuclear retinoid receptors and the transcription ofretinoid-target genes. Gene 328:1–16.

18. Vernet N, et al. (2006) Retinoic acid metabolism and signaling pathways in the adultand developing mouse testis. Endocrinology 147(1):96–110.

19. Jörnvall H, et al. (1995) Short-chain dehydrogenases/reductases (SDR). Biochemistry34(18):6003–6013.

20. Parés X, Farrés J, Kedishvili N, Duester G (2008) Medium- and short-chain dehydrogenase/reductase gene and protein families: Medium-chain and short-chain dehydrogenases/reductases in retinoid metabolism. Cell Mol Life Sci 65(24):3936–3949.

21. Rhinn M, Dollé P (2012) Retinoic acid signalling during development. Development139(5):843–858.

22. Duester G (2008) Retinoic acid synthesis and signaling during early organogenesis.Cell 134(6):921–931.

23. Niederreither K, Subbarayan V, Dollé P, Chambon P (1999) Embryonic retinoic acidsynthesis is essential for early mouse post-implantation development. Nat Genet21(4):444–448.

24. Rhinn M, Schuhbaur B, Niederreither K, Dollé P (2011) Involvement of retinol de-hydrogenase 10 in embryonic patterning and rescue of its loss of function by maternalretinaldehyde treatment. Proc Natl Acad Sci USA 108(40):16687–16692.

25. Sandell LL, Lynn ML, Inman KE, McDowell W, Trainor PA (2012) RDH10 oxidationof vitamin A is a critical control step in synthesis of retinoic acid during mouseembryogenesis. PLoS ONE 7(2):e30698.

26. Sandell LL, et al. (2007) RDH10 is essential for synthesis of embryonic retinoic acid andis required for limb, craniofacial, and organ development. Genes Dev 21(9):1113–1124.

27. Holdcraft RW, Braun RE (2004) Androgen receptor function is required in Sertoli cellsfor the terminal differentiation of haploid spermatids. Development 131(2):459–467.

28. Sadate-Ngatchou PI, Payne CJ, Dearth AT, Braun RE (2008) Cre recombinase activityspecific to postnatal, premeiotic male germ cells in transgenic mice. Genesis 46(12):738–742.

29. Muzumdar MD, Tasic B, Miyamichi K, Li L, Luo L (2007) A global double-fluorescentCre reporter mouse. Genesis 45(9):593–605.

30. Rogakou EP, Pilch DR, Orr AH, Ivanova VS, Bonner WM (1998) DNA double-strandedbreaks induce histone H2AX phosphorylation on serine 139. J Biol Chem 273(10):5858–5868.

31. Enders GC, May JJ, 2nd (1994) Developmentally regulated expression of a mousegerm cell nuclear antigen examined from embryonic day 11 to adult in male andfemale mice. Dev Biol 163(2):331–340.

32. Romanienko PJ, Camerini-Otero RD (2000) The mouse Spo11 gene is required formeiotic chromosome synapsis. Mol Cell 6(5):975–987.

33. Yoshida K, et al. (1998) The mouse RecA-like gene Dmc1 is required for homologouschromosome synapsis during meiosis. Mol Cell 1(5):707–718.

34. Di Carlo AD, Travia G, De Felici M (2000) The meiotic-specific synaptonemal complexprotein SCP3 is expressed by female and male primordial germ cells of the mouseembryo. Int J Dev Biol 44(2):241–244.

35. Morais da Silva S, et al. (1996) Sox9 expression during gonadal development impliesa conserved role for the gene in testis differentiation in mammals and birds. NatGenet 14(1):62–68.

36. Rossant J, Zirngibl R, Cado D, Shago M, Giguère V (1991) Expression of a retinoic acidresponse element-hsplacZ transgene defines specific domains of transcriptional ac-tivity during mouse embryogenesis. Genes Dev 5(8):1333–1344.

37. Tong MH, Mitchell DA, McGowan SD, Evanoff R, Griswold MD (2012) Two miRNAclusters, Mir-17-92 (Mirc1) and Mir-106b-25 (Mirc3), are involved in the regulation ofspermatogonial differentiation in mice. Biol Reprod 86(3):72.

38. Tong MH, Mitchell D, Evanoff R, Griswold MD (2011) Expression of Mirlet7 familymicroRNAs in response to retinoic acid-induced spermatogonial differentiation inmice. Biol Reprod 85(1):189–197.

39. Clermont Y (1972) Kinetics of spermatogenesis in mammals: Seminiferous epitheliumcycle and spermatogonial renewal. Physiol Rev 52(1):198–236.

40. Chen H, Namkung MJ, Juchau MR (1995) Biotransformation of all-trans-retinol andall-trans-retinal to all-trans-retinoic acid in rat conceptal homogenates. BiochemPharmacol 50(8):1257–1264.

41. Deltour L, Haselbeck RJ, Ang HL, Duester G (1997) Localization of class I and class IValcohol dehydrogenases in mouse testis and epididymis: Potential retinol dehydrogenasesfor endogenous retinoic acid synthesis. Biol Reprod 56(1):102–109.

42. Kumar S, Sandell LL, Trainor PA, Koentgen F, Duester G (2012) Alcohol and aldehydedehydrogenases: Retinoid metabolic effects in mouse knockout models. BiochimBiophys Acta 1821(1):198–205.

43. Paik J, et al. (2001) Expression and characterization of a murine enzyme able to cleavebeta-carotene: The formation of retinoids. J Biol Chem 276(34):32160–32168.

44. Rodríguez CI, et al. (2000) High-efficiency deleter mice show that FLPe is an alternativeto Cre-loxP. Nat Genet 25(2):139–140.

45. Russell LD, Ettlin RA, Sinha-Hakim AP, Clegg ED, eds (1990) Histological and Histo-pathological Evaluation of the Testis (Cache River Press, Clearwater, FL).

46. Bianchi M, Tiglao XV (1976) Frequency of the various stages of the seminiferousepithelium in different strains of male mice. Experientia 32(4):503–504.

47. Siiteri JE, Karl AF, Linder CC, Griswold MD (1992) Testicular synchrony: Evaluationand analysis of different protocols. Biol Reprod 46(2):284–289.

548 | www.pnas.org/cgi/doi/10.1073/pnas.1214883110 Tong et al.

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