Proc. Nati. Acad. Sci. USA Vol. 84, pp. 1600-1604, March 1987 Developmental Biology Expression of the proopiomelanocortin gene is developmentally regulated and affected by germ cells in the male mouse reproductive system (testis/gene expression/cellular interactions/opioid peptides/paracrine regulation) ELENA GIZANG-GINSBERG AND DEBRA J. WOLGEMUTH* Department of Genetics and Development and The Center for Reproductive Sciences, Columbia University, College of Physicians and Surgeons, 630 West 168th Street, New York, NY 10032 Communicated by Seymour Lieberman, November 10, 1986

ABSTRACT Proopiomelanocortin (POMC), a major pitu- Cellular differentiation occurs at discrete timepoints during itary product, is also present in the adult mouse testis. We have the development of the mammalian testis, resulting in a shown previously that POMC mRNAs are most abundant in a complex tissue composed of both hormone-producing and subpopulation ofLeydig cells associated with tubules in specific hormone-responsive cells. The germ and somatic cells are stages ofthe cycle ofthe seminiferous epithelium. In the present influenced by hormonal changes during development. study, we examined the expression of the gene encoding POMC POMC-related peptides have been reported to be present in during testicular development and in other tissues of the male the fetal testis and adult testis and at more reduced levels in reproductive system. We also analyzed the effects of cellular the prepuberal testis (6). Regulation at either the transcrip- interactions on POMC gene expression in the testis. Blot- tional or translational level could account for the different hybridization analysis revealed that POMC transcripts of -800 levels of POMC-like peptides observed. nucleotides were present in enriched populations of meiotic POMC-derived peptides have also been observed in other prophase spermatocytes and in caput epididymis but were regions ofthe male reproductive tract, notably the epididymis absent in cauda epididymis and vas deferens. POMC tran- (2). A recent study has provided evidence for local synthesis scripts were present in fetal testis (day 17 of gestation to ofPOMC within the rat epididymis (5). The epididymis is also newborn), could not be detected in prepuberal testis (days 7-8 a complex tissue with unique protein synthetic patterns in the postpartum), but reappeared in the adult testis. No difference caput and cauda regions. The distribution of POMC tran- in the size or abundance ofPOMC transcripts was seen in testes scripts within these regions is not known. from mouse mutant strains in which spermatogenesis is arrest- To address these various points, in the present study we ed in early spermiogenesis. In contrast, POMC transcripts have: (i) determined if the expression of POMC transcripts is were virtually undetectable in testes that are devoid of germ restricted to testicular Leydig cells within the male repro- cells. These results emphasize the importance of interactions ductive system, (ii) examined the pattern of POMC mRNA between germ cells and interstitial cells and the regulation of expression during development of the mouse testis, and (iii) the POMC gene in the mammalian testis. determined if interactions between interstitial cells and cells contained within seminiferous tubules are important in the regulation of POMC expression, and ultimately in its func- The proopiomelanocortin (POMC) gene codes for the pre- in the cursorproteinforneuropeptides such as ,-endorphin, adreno- tion, testis. corticotropin, and melanocyte-stimulating hormones in the MATERIALS AND METHODS pituitary. Immunocytochemical studies have demonstrated Source of Tissues and Cell Populations. Swiss Webster mice the presence of POMC-derived peptides in extrapituitary (Camm, Inc., Wayne, NJ) were the source of tissues or cell tissues, in particular, the mammalian testis (1, 2). We and populations for all experiments except those using mouse others have shown that these peptides probably result from mutant strains. Adult tissues were obtained from mice aged local synthesis, since POMC transcripts are also present in 60 days or older; neonatal testes were from animals at days testicular cells (3-5). POMC transcripts exist as a heteroge- 7-8 of postnatal development. Embryonic testes were dis- neous mRNA species of "-800 nucleotides (nt) in the testis sected from fetuses of pregnant females at day 17 to day 20 and are less abundant than the 1150-nt transcript observed in of gestation or from day 1 newborns (day of vaginal plug is the pituitary. designated day 0 of gestation). Enriched spermatogenic cell Our previous in situ results demonstrated that POMC populations were obtained by separation procedures de- transcripts in the mammalian testis are most abundant in a scribed by Wolgemuth et al. (7). Homozygous and hetero- subpopulation of somatic Leydig cells that are present in zygous littermates of quaking (B6C3Fe-a/a-qk) and interstitial regions associated with discrete tubule stages of atrichosis (ATEB/Le a/a dat/deb) strains were obtained the seminiferous Occasional from The Jackson Laboratory. Testes from mutant strains cycling epithelium (4). labeling were fixed in Bouin's fixative, embedded in paraffin, sec- of spermatogonia and spermatocytes within the adjacent tioned, stained with hematoxylin and eosin, and visualized by tubules was also seen, suggesting that some proportion of light microscopy to confirm the presence or absence of germ POMC transcripts may result from expression in germ cells. cells. These results also suggest that the expression and possibly Sources of Probes. The following probes were used: (i) the function of POMC in the testis may be influenced by pMKSU-16, a 923-base-pair (bp) mouse POMC cDNA, interactions between interstitial cells and cells within the containing the entire POMC coding sequence and additional seminiferous epithelium. flanking sequences (8); (ii) pAl, a 2.0-kilobase (kb) chicken

The publication costs of this article were defrayed in part by page charge Abbreviations: POMC, proopiomelanocortin; FT RNA, flow- payment. This article must therefore be hereby marked "advertisement" through RNA; nt, nucleotide(s). in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed.

Downloaded by guest on September 23, 2021 1600 Developmental Biology: Gizang-Ginsberg and Wolgemuth Proc. Natl. Acad Sci. USA 84 (1987) 1601 p-actin cDNA (9); and (iii) pabl sub3, a v-abl-specific probe A B (10). Plasmid and insert preparations were performed as described by Maniatis et al. (11). Inserts were labeled by 12345678 1 2 3 4 5 6 7 8 nick-translation (12) using all four [32P]dNTPs, yielding specific activities of 1-4 x 108 cpm/gg. RNA Isolation and Blot-Hybridization Analysis. Cells and tissues were frozen in liquid nitrogen, and RNA was isolated by using the lithium chloride precipitation method (13). Poly(A)+ RNA was selected by oligo(dT)-cellulose chroma- tography (Collaborative Research, Waltham, MA, type 3; ref. 14). RNA samples were electrophoresed on a denaturing 1.0% agarose/2.2 M formaldehyde gel, blotted onto nitrocel- 28S - lulose (11), and baked for 3 hr at 80'C. Prehybridizations with sonicated salmon sperm DNA and hybridizations to 32p_ labeled inserts in the presence of dextran sulfate were 18S - performed essentially as described by Wahl et al. (15). Washing conditions were exactly those as described (4). Exposure to x-ray film was at -70'C (1 day to 3 -, - 800 nt weeks). 0 . 4r _~- RESULTS Identification of POMC Transcripts in Enriched Testicular Cell Populations and Other Regions of the Male Reproductive Tract. POMC transcripts have occasionally been observed in germ cells by in situ hybridization analysis (4). Enriched FIG. 1. Blot-hybridization analysis of POMC transcripts in en- populations of spermatogenic cells were obtained to confirm riched germ-cell populations isolated from mature mouse testis. (A) this result by blot-hybridization analysis. Enrichments of RNA samples were electrophoresed and stained with ethidium bromide. (B) The separated RNAs were transferred to nitrocellulose, meiotic prophase spermatocytes (primarily pachytene: -78% hybridized with [32P]pMKSU-16 insert, and autoradiographed (ex- pure), early spermatids (postmeiotic: -85% pure), and re- posure, 2 weeks). Lanes: 1 and 2, poly(A)+ RNA (1.4 ,g) and FT sidual bodies and cytoplasmic fragments (anucleate cell RNA (30 Mg), respectively, from residual bodies and cytoplasmic fragments extruded from elongated spermatids: -95% pure) fragments; 3 and 4, poly(A)+ RNA (4 ,g) and FT RNA (30 ug), were obtained in these particular experiments (7). Leydig cell respectively, from early spermatids; 5 and 6, poly(A)+ RNA (4 Mg) contamination of each population was -4.0%, 1.0%, and and F RNA (30 ,g), respectively, from meiotic prophase sperma- 0.5%, respectively. RNAs were isolated, poly(A)-selected, tocytes; 7 and 8, poly(A)+ RNA (8 Mg) and FT RNA (30 Mg), and probed with the [32P]pMKSU-16 insert. POMC mRNAs respectively, from total testis. Molecular weights, determined from -800 nt long were clearly detected in poly(A)+ RNA from 18S and 28S ribosomal RNAs, are indicated and were used as size standards for all analyses. The faint higher molecular weight band (*) total testis and meiotic prophase spermatocytes (Fig. 1B, is the result of previous hybridization with a testis-specific homeo- lanes 7 and 5) and to a lesser degree in early spermatids (Fig. box containing cDNA, pHBT-1 (16). 1B, lane 3). POMC transcripts were slightly enriched in meiotic spermatocytes compared to total testis RNA: the signal observed in meiotic prophase spermatocyte RNA (Fig. RNAs were also isolated from caput and cauda epididymis 1B, lane 5; 4 ,g) was of equal intensity to that observed with and vas deferens to determine if POMC is synthesized in twice the amount of total testis poly(A)+ RNA (Fig. 1B, lane these tissues. POMC transcripts (-800 nt) were seen in 7; 8 ,ug). No transcripts were observed in either poly(A)+ poly(A)+ RNA from caput epididymis (Fig. 2B, lane 4), RNA from residual bodies and cytoplasmic fragments (Fig. though much less abundantly than in the testis (Fig. 2B, lane 1B, lane 1) or from flow-through (FT) RNA [poly(A)- RNA; 2). No transcripts were detected in poly(A)+ RNA from Fig. 1B, lanes 2, 4, 6, and 8]. A very low level of POMC cauda epididymis and vas deferens (Fig. 2B, lane 6). The blot transcripts was revealed by using higher concentrations of was washed and rehybridized with a probe recognizing the cytoplasmic fragment and residual body RNA (5 ,g; data not c-abl protooncogene transcripts as a positive control for shown). RNA integrity (17). Both somatic c-abl transcripts as well as A B C

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-.0kb a -6.2kb 28S -4.7 k b FIG. 2. Blot-hybridization analysis of testicular .5, POMC transcripts in the male reproductive tract. (A) RNA samples were electrophoresed and stained t8S with ethidium bromide. (B) Autoradiograph of hy- bridization with [32P]pMKSU-16 insert (11-day ex- - - BOOnt posure). (C) Autoradiograph after the blot was washed and rebybridized with a control probe, [32P]pabl sub3 insert (16-day exposure). Lanes: 1 and 2, FT RNA (20 Mg) and poly(A)+ RNA (3 Mg), respectively, from mature testes; 3 and 4, FT RNA (20 Mg) and poly(A)+ RNA (3 Mg), respectively, from caput epididymis; 5 and 6, FT RNA (20 ug) and poly(A)+ RNA (3 Mg), respectively, from cauda epididymis and vas deferens. Downloaded by guest on September 23, 2021 1602 Developmental Biology: Gizang-Ginsberg and Wolgemuth Proc. Natl. Acad. Sci. USA 84 (1987) A B C

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FIG. 3. POMC transcripts during mouse testicular development. (A) RNA samples were electrophoresed 0 Ask -2.3kb and stained with ethidium bromide. (B) Autoradi- -'W -1.65kb ograph after blot hybridization using the [32P]pMKSU- 16 insert (exposure, 11 days). (C) Autoradiograph after the blot was washed and rehybridized with a /3- [32P]actin probe as a positive control (exposure, 3 days). Lanes: 1 and 2, FT RNA (20 ptg) and poly(A)+ RNA (3.8 gig), respectively, from mature testes; 3 and 4, FT RNA (20 ,ug) and poly(A)4 RNA (3.8 pg), respectively, from day 7-8 neonatal testes; 5 and 6, FT RNA (20 jzg) and poly(A)+ RNA (3.8 jug), respectively, from day 17 of gestation-to-newborn testes. the abundant 4.7-kb testis transcript were easily detected Poly (A)' RNA from qk/qk, qk/+ and +/+ testes was (Fig. 2C). analyzed by blot hybridization. The -800-nt band was Expression of POMC Transcripts During Mouse Testicular detected in similar abundance in all three poly(A)+ RNA Development. Testicular differentiation begins at -day 12 of samples (see Fig. 5B; lanes 2, 4, and 6). No transcripts were gestational life in the mouse. Differentiation of the germinal observed in poly(A)- RNAs (FT RNA; Fig. 5B, lanes 1, 3, and somatic components continues during postnatal life until and 5), nor were there any other sized transcripts in poly(A)+ -day 28, when testicular maturation is completed (18, 19). RNA, suggesting that the presence of later-stage spermato- Testes from animals of gestational day 17 to newborns genic cells is not required for regulating POMC expression. contain primitive germ cells, whereas germ cells from day 7-8 We then asked if interactions between interstitial cells and neonates are composed of premeiotic cells. Both embryonic germ cells per se play a role in influencing POMC expression and neonatal testes contain the full complement of somatic cells found in the adult testis, including Leydig, Sertoli, in the testis. The atrichosis mutant, a germ cell-deficient peritubular, and macrophage cells. strain of mice, was used to address this question. Atrichosis POMC mRNAs (-800 nt) were present in poly(A)+ RNA (at) is a single recessive mutation located on chromosome 10 from embryonic day 17-newborn testes, though at a greatly (23). Homozygotes (at/at) are phenotypically identifiable by reduced level as compared to the transcripts observed in their sparsely distributed hair, are sterile, and have abnor- poly(A)I RNA from mature testes (Fig. 3B, lanes 2 and 6). No mally small gonads that are virtually devoid of germ cells. transcripts were detected in poly(A)+ RNA from immature Fig. 4 A and B compare the histology of testes from an at/at day 7-8 testes (Fig. 3B, lane 4), even with higher concentra- tions ofRNA (5 ,Ag; data not shown). As a positive control for RNA integrity, the same blot was rehybridized with /3actin. Two ,/3actin transcripts were easily visualized in adult but not neonatal or embryonic testes (Fig. 3C), consistent with previous results showing that a somatic 2.3-kb transcript and agerm cell-specific 1.65-kb transcript are characteristic ofthe adult testis (C. Ponzetto and D.J.W., unpublished observa- tions; ref. 20). POMC mRNA Expression in Mutant Strains Defective in Spermatogenesis. We have observed (4) localization ofPOMC mRNAs to a subset of Leydig cells associated with discrete stages ofthe cycling seminiferous epithelium-namely, those containing germ cells in later stages of spermiogenesis. The quaking mouse mutant strain provides an interesting model for examining the significance of this relationship. Quaking (qk) is an autosomal recessive mutation resulting in pleiotropic effects, including azoospermia in homozygous (qk/qk) male mice (21). Homozygotes are identified by their involuntary quivering. qk/qk mice exhibit abnormalities in the formation of sperm heads and axonemes during step 8 of spermiogenesis (for stages of spermiogenesis, see ref. 22). These abnormalities may contribute to the appearance of "fragile" spermatids and to the absence of all spermatozoa. Leydig cells from homozygotes (qk/qk) and their heterozy- gous normal littermates (qk/+) exhibit normal steroidogenic activity, as assessed by their ability to secrete testosterone.t Nuclear volumes of Leydig cells and germ cells in earlier stages of spermatogenesis are also normal. FIG. 4. Light micrographs of seminiferous tubules from at/at (A) and at/+ (B) adult mouse testes. Testes were dissected, placed in tChubb, C., 67th Annual Meeting of the Endocrine Society, June Bouin's fixative, sectioned, and stained with hematoxylin and eosin. 19-21, 1985, Baltimore, MD, p. 73 (abstr.). (x 130.) Downloaded by guest on September 23, 2021 Developmental Biology: Gizang-Ginsberg and Wolgemuth Proc. Natl. Acad. Sci. USA 84 (1987) 1603 A B C

1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10

...... I Ia 28S

- 2.3kb 1 8S is 49 1 is 40 fe 4 I - 1.65 kb

--800nt I *

FIG. 5. POMC transcripts in adult testes from mouse strains defective in spermatogenesis. (A) RNA samples were electrophoresed and stained with ethidium bromide. (B) Autoradiograph after blot hybridization with [32P]pMKSU-16 insert (exposure, 3 days). (C) Autoradiograph after the blot was washed and rehybridized with 1p[32P]actin probe as a positive control (exposure, 3 days). Lanes: 1 and 2, FT RNA (25 Mg) and poly(A)4 RNA (10 Mg), respectively, from normal +/+ testes; 3 and 4, FT RNA (25 Mg) and poly(A)+ RNA (10 Mg), respectively, from normal qk/+ testes; 5 and 6, FT RNA (25 Mg) and poly(A)+ RNA (10 Mg), respectively, from mutant qk/qk testes; 7 and 8, FT RNA (25 Mg) and poly(A)+ RNA (10 ,ug), respectively, from normal at/+ and +/+ testes; 9 and 10, FT RNA (25 Mg) and poly(A)+ RNA (10 Mig), respectively, from mutant at/at testes. mouse and its normal littermate. Leydig cells from the we further examined this result by preparing enriched pop- homozygous mutant testes exhibit a decreased nuclear mass ulations of spermatogenic cells. Our detection of POMC and increased cytoplasmic volume, a common feature of transcripts in the spermatocyte fraction suggests that these testes containing degenerate germ cells (24). However, POMC-derived proteins are synthesized in the cell types in Chubb and Nolan (24) have demonstrated that the Leydig which they are found. cells of at/at mice are steroidogenically normal, secreting We do not believe that the POMC transcripts seen in the nine steroids (including testosterone) at rates comparable to enriched spermatogenic cell populations are the result of con- their normal littermates. Maturation ofSertoli cells within the taminating Leydig cells because (0) the presence of POMC seminiferous epithelium is essentially normal (25). transcripts in the spermatocyte fraction confirms our previous RNA was isolated from at/at, at/+, and +/+ testes and results at the in situ level, and (it) Leydig cells are a minor was analyzed by blot hybridization (Fig. 5). POMC tran- contaminant (-4.0-0.5%) of these enriched germ-cell popula- scripts (-800 nt) were easily detected and were present in tions. We consider it unlikely, but cannot rule out the possibil- equal abundance in poly(A)+ RNA from +/+ and at/+ mice ity, that the subpopulation of Leydig cells that contain POMC (Fig. 5B, lanes 2 and 8). In contrast, POMC transcripts were mRNAs in highest abundance (t2.0%o ofall interstitial cells; ref. not detected in poly(A)+ RNA from at/at adult testis (Fig. 4) are preferentially selected for in the enrichment of the 5B, lane 10). The blot was rehybridized with the ,/-actin probe spermatocyte population. Interestingly, Kilpatrick and Millette as a positive control (Fig. 5C). The somatic 2.3-kb transcript (27) recently demonstrated that minor Leydig cell contamina- was easily discernible in poly(A)+ RNAs from all adult testes tion of enriched testicular cell populations, obtained by similar analyzed (Fig. 5C, lanes lanes 2, 4, 6, 8, and 10). The separation procedures, could not account for the level of testis-specific 1.65-kb transcript could be seen in RNA from expression of the proenkephalin gene observed. +/+, qk/+, qk/qk, and at/+ testes (lanes 2, 4, 6, and 8) and (3-Endorphin-like material has been localized immunocy- not in the germ cell-deficient at/at mice (lane 10), consistent tochemically in the epithelium of mouse caput epididymis with previous results demonstrating its association with germ and, to a lesser extent, in the cauda epididymis and vas cells (C. Ponzetto and D.J.W., unpublished observations; deferens (2). POMC transcripts have been observed in RNAs ref. 20). isolated from total rat epididymis (5), but no distinction was made as to expression in discrete epididymal regions or in the DISCUSSION vas deferens. POMC transcripts of the testicular-size class These results provide new insight into the importance of germ rather than the pituitary-size class were detected in the cell-interstitial cell interaction in the regulation of POMC gene mouse caput epididymis but not in cauda epididymis and vas expression in the mammalian testis. We confirm and extend our deferens. These observations suggest that POMC production previous observations and those of others by examining the is the result of local synthesis in the caput epididymis. The distribution ofPOMC transcripts in enriched spermatogenic cell precise cell type expressing POMC transcripts within the populations during development ofthe testis and in othertissues epididymis is unknown. Although the inability to detect within the male reproductive system. POMC transcripts in the cauda epididymis and the vas Cheng et al. (26) recently demonstrated immunoreactivity deferens may be due to limits in the sensitivity of blot- for the a-N-acetylated form ofendorphin in spermatocytes as hybridization analysis, we can conclude that (0) POMC well as in spermatogonia. We had noted previously a low transcripts are expressed in the epididymis at a reduced level level of labeling of spermatogenic cells by in situ hybridiza- relative to expression in the testis, and (ii) expression in the tion with a POMC cDNA as a probe (4). In the present study, epididymis is most abundant in the caput. Downloaded by guest on September 23, 2021 1604 Developmental Biology: Gizang-Ginsberg and Wolgemuth Proc. Natl. Acad. Sci. USA 84 (1987) Changes in the levels of POMC-like peptides during tes- gene in the testis. Since POMG transcripts were not detected ticular development have been observed (6). Such alterations in testis of at/at mice, the possibility that germ-cell produc- could result from regulation at either the transcriptional or tion may be necessary for certain aspects of Leydig-cell translational level. Our results show that the levels of POMC development and function must be considered. The impor- mRNAs parallel the changes seen at the protein level. POMC tance of cell-cell interactions suggests that regulation of the transcripts were detected in gestational day 17-newborn POM gene in the testis may involve paracrine effects. testis, although the abundance ofPOMC mRNAs was greatly We thank Drs. H. Calvin, R. Palmiter, and J. Roberts and our reduced compared to the adult testis. In day 7-8 neonatal laboratory colleagues for critical reading of this manuscript; G. testis, the level of POMC transcripts, if present, was below Grills, S. Drosinos, S. deGolia, and L. Chou for help in photography the limits of sensitivity of blot-hybridization analysis. The and typing; and Drs. E. Herbert, D. Cleveland, and S. Goff for their stages in which POMC expression is detected exactly parallel generous gifts of plasmids. This work was supported by grants from prenatal and postnatal peaks of Leydig-cell differentiation in the National Institutes of Health, HD 05077 (to D.J.W.) and AM the mouse (28, 29). 07330 (to E.G.G.) and a grant from the I.T. Hirschl Trust (to D.J.W.). Our previous work suggested that cell-cell interactions 1. Margioris, A. N., Liotta, A. S., Vaudry, H., Bardin, C. W. & may play a role in influencing testicular POMC expression, Krieger, D. T. (1983) Endocrinology 113, 663-671. since POMC transcripts were most abundant in Leydig cells 2. Tsong, S.-D., Phillips, D. M., Halmi, N., Krieger, D. & in association with stages IX-XII of the seminiferous epithe- Bardin, C. W. (1982) Biol. Reprod. 27, 755-764. lium (4). Stage IX-XII tubules contain later stages of sper- 3. Pintar, J. E., Schachter, B. S., Herman, A. B., Durgerian, S. matogenic cells in addition to a precise arrangement ofearlier & Krieger, D. T. (1984) Science 225, 632-634. The mutant strain enabled us 4. Gizang-Ginsberg, E. & Wolgemuth, D. J. (1985) Dev. Biol. germ-cell types. quaking mouse 111, 293-305. to examine the relationship between POMC expression and 5. Chen, C.-L. C., Mather, J. P., Morris, P. L. & Bardin, C. W. the presence oflater germ-cell stages, since cells correspond- (1984) Proc. Nadl. Acad. Sci. USA 81, 5672-5675. ing to these developmental stages are absent in qk/qk mice. 6. Shaha, C., Liotta, A. S., Krieger, D. T. & Bardin, C. W. The abundance of POMC transcripts appeared to be similar (1984) Endocrinology 114, 1584-1591. in normal adult testes, qk/qk mutant testes, and testes from 7. Wolgemuth, D. J., Gizang-Ginsberg, E., Engelmyer, E., normal qk/+ littermates. This suggests that the appearance Gavin, B. J. & Ponzetto, C. (1985) Gamete Res. 12, 1-10. of testicular POMC transcripts is not dependent upon the 8. Uhler, M. & Herbert, E. (1983) J. Biol. Chem. 258, 257-261. presence of cells in later stages of spermiogenesis. However, 9. Cleveland, D. W., Lopata, M. A., MacDonald, R. J., Cowan, cell N. J., Rutter, W. J. & Kirschner, M. W. (1980) Cell 20, 95-105. it will be important to consider the effects of other types, 10. Wang, J. Y. J., Ledley, F., Goff, S., Lee, R., Groner, Y. & such as meiotic prophase spermatocytes, which are present Baltimore, D. (1984) Cell 36, 349-356. in stage IX-XII tubules. 11. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular The -800-nt POMC mRNA was undetectable in homozy- Cloning: A Laboratory Manual (Cold Spring Harbor Labora- gous atrichosis males, which have testes completely devoid tory, Cold Spring Harbor, NY). ofgerm cells. Although we have shown that some proportion 12. Weinstock, R., Sweet, R., Weiss, M., Cedar, H. & Axel, R. of testicular POMC expression is the result of various (1978) Proc. Natl. Acad. Sci. USA 75, 1299-1303. germ-cell types, particularly meiotic spermatocytes, their 13. Cathala, G., Savouret, J.-F., Mendez, B., West, B. L., Karin, absence alone in at/at testes cannot account for the drastic M., Martial, J. A. & Baxter, J. D. (1983) DNA 2, 329-335. 14. Aviv, H. & Leder, P. (1972) Proc. Natl. Acad. Sci. USA 69, decrease of message observed. Meiotic spermatocytes con- 1408-1412. stitute 18% of cells in the intact adult mouse testis (30) and 15. Wahl, G. M., Stern, M. & Stark, G. R. (1979) Proc. Natl. =78% of the enriched population. Theoretically, we would Acad. Sci. USA 76, 3683-3687. expect at least a 4- to 5-fold enrichment of POMC transcripts 16. Wolgemuth, D. J., Engelmyer, E., Duggal, R. N., Gizang- in the spermatocyte fraction. RNAs from the enriched Ginsberg, E., Mutter, G. L., Ponzetto, C., Viviano, C. & meiotic prophase spermatocyte fraction showed at most a Zakeri, Z. F. (1986) EMBO J. 5, 1229-1235. 2-fold greater signal compared to RNAs from intact testis. 17. Ponzetto, C. & Wolgemuth, D. J. (1985) Mol. Cell Biol. 5, The ratio of Leydig cells to other cell types is actually 1791-1794. increased, since at/at testes contain no germ cells. There- 18. Nebel, B. R., Amarose, A. P. & Hackett, E. M. (1961) Sci- ence 134, 832-833. fore, if POMC expression is unaffected by interactions 19. Bellve, A. R., Cavicchia, J. C., Millette, C. F., O'Brien, D. A., between interstitial cells and germ cells within the seminifer- Bhatnagar, Y. N. & Dym, M. (1977) J. Cell Biol. 74, 68-85. ous tubules, an increase in the level ofPOMC mRNAs should 20. Waters, S. H., Distel, R. J. & Hecht, N. B. (1985) Mol. Cell perhaps have been detected. This was not the case. Biol. 5, 1649-1654. Leydig cells have been shown to be a major source of 21. Bennett, W. I., Gall, A. M., Southard, J. L. & Sidman, R. L. POMC transcripts in the mammalian testis in previous studies (1971) Biol. Reprod. 5, 30-58. (3-5). Although Leydig cells in at/at mice have been shown 22. Oakberg, E. F. (1956) Am. J. Anat. 99, 391-409. to be steroidogenically normal (24), our results show that 23. Hummel, K. P. (1964) Mouse News Lett. 34, 31-32. they are not normal with respect to POMC expression. 24. Chubb, C. & Nolan, C. (1984) Ann. N.Y. Acad. Sci. 438, of 519-522. Sertoli cells are present within the seminiferous epithelium 25. Handel, M. A. & Eppig, J. J. (1979) Biol. Reprod. 20, 1031-1038. at/at mice and exhibit essentially normal maturation prop- 26. Cheng, M. C., Clements, J. A., Smith, A. I., Lolait, S. J. & erties (25). This suggests that germ-cell production is not a Funder, J. W. (1984) J. Clin. Invest. 75, 832-835. prerequisite for structural differentiation of Sertoli cells and 27. Kilpatrick, D. L. & Millette, C. F. (1986) Proc. Natl. Acad. that the presence of Sertoli cells alone is not sufficient for Sci. USA 83, 5015-5018. normal POMC expression. 28. Russo, J. (1971) Anat. Rec. 170, 343-356. 29. Russo, J. & DeRosas, J. C. (1971) Am. J. Anat. 130, 461-480. Various roles for POMC in the mammalian testis have been 30. Meistrich, M. L., Bruce, W. R. & Clermont, Y. (1973) Exp. postulated. Neuropeptides derived from the POMC gene Cell Res. 79, 213-227. have been shown in vitro to stimulate cAMP accumulation 31. Bardin, C. W., Shaha, C., Mather, J. P., Salomon, Y., and affect the growth rate of Sertoli cells as well as to Margioris, A. N., Liotta, A. S., Gerendai, I., Chen, C-L. C. & facilitate testosterone secretion from Leydig cells (31-33). Krieger, D. (1984) Ann. N. Y. Acad. Sci. 438, 346-364. 32. Gerendai, I., Shaha, C., Thau, R. & Bardin, C. W. (1984) Although no direct function in germ-cell development has yet Endocrinology 15, 1645-1647. been ascribed for POMC products, our results demonstrate 33. Chen, C.-L. C., Chang, C.-C., Krieger, D. T. & Bardin, C. W. that germ cells are critical for the regulation of the POMC (1986) Endocrinology 118, 2382-2389. Downloaded by guest on September 23, 2021