Expression and Regulation of a Proenkephalin ,&Galactosidase Fusion Gene in the Reproductive System of Transgenic Mice

David Borsook, Haim Karl Herrup*, Michael

Rosen, Michael Collard, Holly Dressler, J. Comb, and Steven E. Hyman

Molecular Neurobiology Laboratory (D.B., H.R., M.C., S.H.) Department of Anesthesia (D.B.) Department of Neurology (D.B., M.Com.) Department of Psychiatry (S.H.) Massachusetts General Hospital Harvard Medical School Boston, Massachusetts 02114 Department of Molecular Virology (HR.) The Hebrew University Hadassah Medical School Jerusalem, Israel Toxicology and Molecular Biology Programs (M.Col.) Utah State University Logan, Utah 84322-5600 Department of Developmental Neurobiology (H.D., K.H.) EK Shriver Center Waltham, Massachusetts 02254

INTRODUCTION

A fusion gene containing 3 kilobases of human proenkephalin 5’-flanking sequences and 1 kilobase of human proenkephalin 3’-flanking sequence and the easily visualized histochemical marker, Escherichia co/i ,&galactosidase, was used to study the function of cis-regulatory elements within the human proenkephalin gene in transgenic mice. Here data are presented on expression and regulation of this fusion gene in the reproductive system of three independent lines of transgenic mice. Within the male reproductive system, the fusion gene is expressed in the proximal epididymis and in developing germinal cells but not in mature or elongating spermatids. In the female reproductive system, the transgene was expressed at low basal levels, but expression was dramatically stimulated in the ovary and oviduct by hormonal stimulation and pregnancy; additionally, expression was induced at the uteroplacental junction in pregnant mice. Taken together these observations suggest that critical sequences for expression and regulation of the proenkephalin gene within the reproductive system are contained within sequences of the construct. (Molecular Endocrinology 6: X02-1512, 1992) 0888-8809/92/l 502-l 512$03.00/O Molecular Endocrinology Copyright 0 1992 by The Endocrine

Major determinantsof cell type-specific and physiologically regulated gene expression are the multiple cisregulatory elements that are arrayed upstream and occasionallydownstreamof the transcriptionalstart site (1). Mutational analyses, performed primarily with cell culture models,have shown that each eukaryotic gene hasa particularcombinationof &-regulatory elements. The nature, number, and spatial arrangementof these elements determine the gene’s unique pattern of expression. These regulatory elementsmay determine the cell types in which the gene is expressed,the times during development at which it is expressed, and the level at which it is expressed both at basal levels and in response to physiological signals. However, for genes such as proenkephalin,which encodes the precursor of the opioid peptides met- and leu-enkephalin, and which displays complex patterns of expression in multiplecell types, cell culture modelsare inadequate. In particular, the proenkephalingene is expressed in many cell types in viva, includingmultiple neuronalcell types, spermatogenic, and epididymal cells that lack good cell culture models. Thus a transgenic mouse model provides the best available method to study molecular mechanismsregulating this gene in the reproductive system.

Society

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1503

Human Proenkephalin Gene Expression

We have previously characterized the function of a CAMP, phorbol ester, and calcium-inducible enhancer within the human proenkephalin gene (2-5). This enhancer consists of multiple overlapping DNA elements (6) extending between nucleotides -110 and -70 with respect to the mRNA cap site. Reporter gene fusion constructs containing up to 193 base pairs (bp) of human proenkephalin 5’-flanking sequence (and therefore containing the enhancer) have been found to express at high levels in many cell types including CV-1, HeLa, and PC12 cells (2, 3). However, these cell types do not express their endogenous proenkephalin gene. It is likely, therefore, that restriction of expression to a limited number of cell types in vivo is determined at least partly by additional regulatory sequences or cell type-specific trans-acting factors. To examine this possibility, we have begun to produce and analyze transgenie mice expressing a DNA fusion construct which contains a histochemically detectable reporter gene, LacZ, encoding Escherichia coli @-galactosidase (pGal). In these initial studies we analyzed three lines of mice in which LacZ expression was directed by wild type human proenkephalin regulatory sequences containing 3 kilobases (kb) of .5’-flanking sequences and 1 kb human proenkephalin 3’-flanking sequences. We report here on the expression of this fusion gene within the male and female reproductive system of the mouse. The rodent reproductive system is an excellent model system in which to study cell type-specific expression of the proenkephalin transgene, because a great deal is known about expression of the endogenous proenkephalin gene in these tissues (7-l 4). In males the gene is expressed in the proximal epididymis (15, 16) and in the testis (7-10, 15, 17). In the testis the expression of a number of genes, including proenkephalin, is developmentally regulated during spermatogenesis (18, 19). Among testicular cell types, the proenkephalin gene is expressed in immature spermatocytes and round spermatids, but not in elongating spermatids (18, 19). In females the proenkephalin gene is expressed within the uterus (10, 12, 13) the ovary (9, 10, 12-14, 20-22) and the oviduct (12). The exact function of proenkephalin gene expression in these tissues remains to be determined, but a putative role in autocrine/paracrine regulation within the reproductive system has been suggested (9, 12-l 4, 16, 22). In support of this hypothesis, proenkephalin gene expression is significantly elevated within the uterine endometrium, predominantly at the implantation site, during pregnancy (12) and is enhanced by estrogens (14, 22). In the male reproductive system, proenkephalin may play a significant role in the maturation of sperm (11, 15, 16). Proenkephalin expressed in spermatogenic cells may act to regulate sperm development (8) while proenkephalin-derived peptides in the mature sperm acrosome have been proposed to function during fertilization (11). In addition, the entry of sperm into the epididymis is thought to induce high-level expression of proenkephalin in principal cells which line the lumen of the epididymal duct

(16).

The 193-bp promoter sequence of the human proenkephalin gene fused to the reporter, chloramphenicol acetyltransferase (CAT), that we have analyzed in tissue culture (2-5), confers expression in the testis (23). However, the CAT reporter does not readily permit histological or cell type-specific analysis of this expression. Zinn et al. (24) have recently reported selective transcription of the rat proenkephalin gene fused to a CAT reporter in the testis of transgenic mice. The rat and mouse proenkephalin genes have been reported to contain a germ cell-specific promoter (25); the human proenkephalin promoter, however, lacks these putative germ cell-specific sequences (6). Nonetheless, our results show that the human proenkephalin transgene is expressed in mice with a pattern similar to that previously described for the endogenous proenkephalin gene, indicating that the single promoter within human proenkephalin gene contains the necessary information for correct expression in the mouse germ cells. Furthermore, the p-Gal reporter gene allows detailed analysis of expression at a histological level.

RESULTS Construction of Human Proenkephalin/B-Gal Gene and Production of Transgenic Animals

Fusion

Figure 1 shows the plasmid pENK-/I-Ga12.3, from which the microinjected sequences were derived. These sequences contain 3 kb human proenkephalin gene 5’flanking sequences, the first exon and intron, the E. coli LacZ transcription unit, and 1 kb human proenkephalin 3’ flanking sequences. Three independent founder lines of mice (ENK 1 .l, 1.2, and 1.3) were produced, all of which expressed the fusion gene within the reproductive system. None of the mice had apparent morphological or behavioral abnormalities. Table 1 summarizes the pattern of expression in the ENK 1.1 line for the male and female reproductive systems compared with the wild type (C57BL/6J) based on analysis of both whole mount and 40-micron sections histochemically stained using the chromogenic substrate of p-Gal, 5-bromo-4-chloro-3-indolyl-B-o-galactopyranoside (XGal). Results for ENK 1.2 and ENK 1.3 were similar. Expression of the Transgene Reproductive System

in the Male

Within the male reproductive system significant levels of transgene expression were observed in testes of all three transgenic lines as determined by X-Gal histochemistry. Figure 2 shows photomicrographs of sections from the testes of wild type (Fig. 2A), juvenile transgenic (P7; Fig. 2B), and adult transgenic mice (Fig. 2, C-E). There was no expression of p-Gal in the testes of adult wild type mice (Fig. 2A). In the adult transgenic testes, high levels of p-Gal expression were present. The staining was observed at the basal/proximal region of the seminiferous tubules, suggesting that sperm cells

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MOL 1504

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1992

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Table

1. Summary

of p-Gal

Staining

in Reproductive Enk 1 .l

Male Adult

Testis

pENKBGAL-2.3 10.5 KB

B,.EL Fig. 1. pENK-p-Ga12.3 To obtain adequate 5’-flanking sequences of the proenkephalin gene, we screened a human cosmid library (a gift of Dr. Werner Lindenmaier, Gesellschaft fur Biotechnologishe Forschung, Braunschweig, Germany). Of 10 positive cosmids, cosmid E.2 contained approximately 10 kb proenkephalin 5’flanking sequence. A 3-kb Xbal to Sac1 fragment derived from this cosmid E.2 was cloned into the backbone provided by pENKAT-12 (5) which contains human proenkephalin 5’-flanking sequences spanning nucleotides -193 to +130 with respect to the mRNA cap site fused to bacterial chloramphenicol acetyl transferase (CAT) and 1 kb human proenkephalin 3’flanking sequences. The 5’-Xbal site was replaced with a unique Not1 site. The CAT transcription cassette was replaced by an E. co/i P-Gal transcription cassette (devoid of any promoter or 3’-flanking sequences) derived from pCHll0 (a gift of Malcom Low, Vollum Institute, Oregon) by converting a Hincll site at the 5’-border of the CAT cassette into a HindIll site. CAT was removed by a Hindlll/BamHI digestion and replaced by a P-Gal HindIll to BamHl cassette. The DNA

sequences for injection were obtained from a Notl-Sphl digestion which

yielded

a 7.6-kb

fragment.

are expressing the transgene rather than the somatic cell types of the tubule (Figs. 2F and 4D). Mature sperm, seen within the lumen of the tubules, do not stain for p-Gal, and no staining is seen in mature sperm isolated from the epididymis. Levels of transgene expression within the seminiferous tubules appeared to be high in some tubules and low or minimal in others. This pattern probably results from different ratios of immature to mature germ ceils within different regions of the seminiferous tubules, consistent with the normal spermatogenie cycle (see Fig. 2, C-D). No p-Gal activity could be detected in the juvenile transgenic male testes examined at stages P7, P14, and P21, further indicating that expression of the gene in the testis is dependent on the stage of germ cell development. Figure 3 shows a Northern analysis for P-Gal mRNA derived from testis and epididymis (see below) of transgenic and wild type animals. E. co/i p-Gal transcripts were detected only in RNA from the testes of transgenic mice. p-Gal mRNA

Epididymis Juvenile (P7, P14, P21) Testis Epididymis Female Ovary” Uterus” Oviducta Female (pregnant) Ovary Uterus Uteroplacental junction Female (PMS + hCG)b Ovary Uterus Oviduct

+++ ++++

No. 9

Organs

Wild type 057BL/6JI

++a

-

-

+/-a

+/-”

+/+/+/-

-

+++ +++ +++

-

+++ +++ +++

-

Relative intensity of P-Gal staining is indicated by -; no staining, +/-; minimal and variable staining or grades of positive staining, ++ to ++++. P7, P14, and P21 indicate age (days) of juvenile males. a See text for details. b See Materials and Methods.

in the testis was expressed as two bands similar to that reported by Forss-Pretter et al. (26) for a neuronspecific enolase-P-Gal fusion. The overall pattern of transgene expression within the these male reproductive tissues corresponds well with previous reports of endogenous proenkephalin gene expression (7-9, 1517), indicating that the transgene responds to the normal developmental and hormonal control mechanisms in the testis. Transgene expression was compared to endogenous proenkephalin mRNA expression by in situ hybridization and Northern blot analysis. In situ hybridization analysis of mRNA for the endogenous proenkephalin gene shows a regional distribution similar to the transgene product (Fig. 4, A and B, and Fig 20). The region of the seminiferous tubules that hybridizes most strongly (arrows indicate borders of tubules) is the region containing the developing germ cells. There is minimal hybridization (no greater than background) in the center of the tubules, where the mature sperm are located, or outside the tubules (Fig. 4, B and C). Immunohistochemistry for p-Gal (Fig. 4D) predominantly stains spermatocytes and round spermatids within the same region of the seminiferous tubules that express the highest levels of endogenous proenkephalin mRNA by in situ hybridization. Figure 5 shows a Northern analysis of p-Gal and proenkephalin mRNA distributions in enriched populations of cells (Leydig cells, total germ cells, spermatocytes, round spermatids, and elongating spermatids) isolated from transgenic testes (see Materials and Methods). The highest levels of p-Gal transcripts occur in spermatocytes and round spermatids, with no

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Human

Proenkephalin

Gene

Expression

1505

Fig. 2. Histological Sections of Mouse Testes Stained for P-Gal Panels A-F show 45-micron histological sections of testes stained for D-Gal. Phase contrast photomicrographs are shown from sections of testes of (A) adult (P49) wild type and (B) juvenile (P7) transgenic mice. Panel C shows a section through the whole testis of an adult (P49) transgenic mouse. Panel D shows a higher power view indicating the distribution of B-Gal staining within the seminiferous tubules. Panel E shows a similar section counter-stained with neutral red. Panel F shows a higher power view of p-Gal staining within the seminiferous tubules.

C El E2 E3 Testis

c

28s

+

18s

C El Epidid.

Fig. 3. Northern Analysis for p-Gal mRNA in Adult Testis and Epididymis from ENK 1 .l, 1.2, and 1.3 Transgenic Lines (El, 2, 3) Compared with Wild Type (C) Mice Twenty micrograms of total mRNA were loaded in each lane. (See Materials and Methods for details.)

consistent with histochemical analyses shown in Figs. 4, A-C, and 2, D-F, respectively. For the endogenous proenkephalin gene, the highest level of mRNA occurs in round spermatids. A lower level was observed in spermatocytes, and little expression was observed in elongating spermatids. This result is similar, although not identical to the p-Gal distribution, and is consistent with the distribution previously reexpression results from

in elongating spermatids, in situ hybridization and

ported for proenkephalin mRNA in the wild type mouse testis (18, 19). The 1.8-kb mRNA for proenkephalin is also a germ cell-specific marker that is larger than the 1.4-kb transcript normally found in somatic cells. The presence of the 1.8-kb transcript in the Leydig cell fraction in Fig. 5 indicates about a 15% contamination of the fraction by germ cells and is similar to contamination estimates made by light microscopy. The /I-Gal mRNA in the Leydig cell fraction is therefore most likely of germ cell origin and not due to expression of the transgene in Leydig cells. The combined results of the above

analysis

indicate

that

the

human

proenkephalin

fusion gene is expressed and developmentally regulated in mouse testis in a manner nearly identical to the endogenous proenkephalin gene. High levels of p-Gal staining were also observed in the epididymis in all three transgenic lines. However, unlike the testis the epididymis contains an endogenous P-Gal-like activity that produces background staining with X-Gal histochemistry. Thus, some staining was observed in the epididymis of the wild type animals (Fig. 6). The high levels of P-Gal staining seen in the epididymis of transgenic animals reflect the sum of expression due to the transgene and to the endogenous pGal-like activity. The epididymis of transgenic animals

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Fig. 4. In Situ Hybridization Sections of Mouse Testis Panels A-C show ,n situ hybndlzatlon for proenkephalln mRNA performed on sections (20 pm) through the mouse testis. Panel A shows a bnghtfleld and panel B a darkfleld photomlcrograph of the same sectlon. Autoradlography was performed for 2 weeks Panel C shows a higher power brIghtfIeld photomlcrqraph for a semlnlferous tubule lndlcatlng the cell types where most of the hybrrdization IS found (I e between the arrows). Note that hybndlzataon over the mature sperm withan the center of the tubule or outside the semlnlferous tubule IS not above background levets Panel D shows the cilstrlbutlon of +Gal-posltlve cells after immunohistochemlstry with a &Gal antibody, positively staining cells are in the same distnbutlon as shown In C The most darkly starning cells are mostly round spermatids (See Rg 5 for Northern analyss and Fig 20 for comparison with J-Gal histochemistry ) In panels A and B the smaN arrows indicate the basal limit of the seminiferous tubules in corresponding sections A and B.

therefore stains far darker than the wild type (not shown). Northern analysis using a P-Gal probe confirmed that the @-Gal-like activity within the wild type epididymis was not due to expression of sequences from the E. co/i LacZ gene or any closely related sequences (see Fig. 3). Figure 6 shows photomicrographs of sections from the proximal and distal epididymis of transgenic (A and 6) and wild type mice (C and D), demonstrating the expected regionally specific expression of the transgene within the epididymis of transgenic mice. High levels of @-Gal staining were present in the proximal epididymis and lower levels in the distal epididymis (16). Expression of the Transgene Reproductive System

in the Female

B-Gal expression in the ovary, uterus, and oviduct of female transgenic mice of the ENK 1 .l line was minimal in some animals and absent in others. Though not rigorously tested, this variation may reflect the stage of the estrous cycle at which the animals were killed since levels of proenkephalin gene expression are known to be cycle dependent (13, 22). Whole mounts of the

ovary, oviduct, and uterus stained with X-Gal are shown in Fig. 7 for wild type (Fig. 7A), transgenic nonpregnant (Fig. 7B). pregnant (Fig. 7C), and hormonally stimulated (Fig. 70) females. @-Gal staining is clearly seen n the ovary, oviduct, and uterus of both the pregnant and hormonally stimulated transgenic females, but not m the untreated, nonpregnant transgenic or wild type females (Fig. 7). More detailed analysis of these changes is shown in photomicrographs of corresponding sectrons of the ovaries. There are high levels of 8Gal expression in the theta cells of preovulatoty follicles and granulosa cells which surround the developing oocyte in both the pregnant (Fig. 8, C-D) and hormonally stimulated animals (see lwaterials and !&thods; not shown) but not in the nonpregnant control animal (Fig. 8, A and B). The pattern of expression IS identical to that previously shown for proenkephalin mRNA in granulosa cells surrounding the oocyte using /fl situ hybridizatron (12). The results indicate that the transgene is under hormonal control (pregnancy) and can be strongly induced by administration of exogenous PMS and human CG (hCG). In pregnant female transgenic mice, high levels of fiGal expression are present at the utero-placental junc-

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Human

Proenkephalin

Gene

Expression

R-Galactosidase

1507

/

Proenkephalin

LTSRE Fig. 5.

Northern Analysis for p-Gal mRNA (Upper) and the Same Blot Reprobed with a Rat Proenkephalin Exons 1 and 2 (Lower) RNA was derived from isolated testicular cells (see Materials and Methods) including Leydig spermatocytes (S), round spermatids (R), and elongating spermatids (E); 0.8 pg total mRNA was loaded and Methods for details.)

tion. Figure 9 shows strong staining at the utero-placental junction in whole mounts of the uterus from pregnant mice killed on the 7th or 12th day of pregnancy. In Fig. 9A, staining was seen in a small, discrete area, but as pregnancy advances, the distribution of staining becomes continuous within the uterus from one utero-placental junction to the next (Fig. 9B). Histological sections (Fig. 9C) through the region shown in Fig. 9A shows intense p-Gal staining in the endometrial wall of the uterus at the uteroplacental junction (Fig. 9, C and D), consistent with what has been reported by in situ hybridization for the endogenous gene (12).

DISCUSSION

Proenkephalin-derivedpeptides have been implicated in a wide variety of important biologicalprocessesboth within the reproductive system and outside it, e.g. spermmaturation(4, 1I), pregnancy (13), seizures(2729), analgesia(30-32) endocrine regulation (33), organogenesis(34, 35) and immunity (36, 37). The sequences within the human proenkephalin gene that regulate expression in responseto CAMP, the protein kinase C pathway, and calcium have been characterized in detail in tissue culture model systems (2-6).

Genomic

Probe

Encoding

cells (L), whole testis (T), in each lane. (See Materials

However, tissue culture models cannot be used to study cell type-specific expression in the reproductive or nervous systems, because of the lack of adequate cell lines.Therefore, we employeda transgenicstrategy to start to analyze the regulatory elementsinvolved in cell type-specific expression. In this study the patterns of expression of a human proenkephalinp-Gal fusion gene in the maleand female reproductive systemof transgenicmicewere examined. Expression of p-Gal, the easily visualized reporter of transgeneexpression, was similarto expressionof the endogenousproenkephalingenein the maleandfemale reproductive systems (7-l 3, 13-l 7, 20-22). In males, high levels of P-Gal activity were observed in spermatocytes and round spermatids, with essentially no expressionin mature spermatidsor somatic(Leydig or Sertoli) cells. No p-Gal expressionwas observed in the testes of immature males(P7, P14, and P21), further indicating that transgene expression was limited to developinggerm cells.In contrast with our observations in vivo in transgenic mice, expression of the proenkephalin gene has been reported in cultures of juvenile rodent Sertoli cells (38) and adult Leydig cells (39). These differences may be due to induction of proenkephalingene expression by the processof cell culture itself. Northern analysisconfirmedthat levelsof both pGal mRNA and endogenousproenkephalinmRNA were

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MOL ENDO. 1992 1508

Vol6 No. 9

Fig. 6. Photomicrographs of 45-Micron Sections Showing Sections of Proximal (A and B) and Distal (C and D) Epididymis of Adult Mice (P42) After X-Gal Histochemistry and Counterstaining with Neutral Red Sections A and C were from an ENK 1 .l transgenic mouse and B and D from a wild type mouse. (See Materials and Methods for details.)

highest In spermatocytes and round spermatids; however, proenkephalin mRNA levels were relatively lower in spermatocytes than p-Gal mRNA levels, suggesting some slightly anomalous expression of the transgene at this early stage of germ cell development. We note, however, that the levels of transgene expression in mouse germ cells appear identical to what we have observed for endogenous proenkephalin gene expression in developing rat germ cells (Collard, M., unpublished observations). In the epididymis, very high levels of transgene expression were observed, particularly in the proximal segment. This is in good agreement with previous studies of the endogenous gene (16). In unstimulated female transgenic mice p-Gal was expressed in granulosa cells surrounding developing oocytes within the ovary, with minimal expression in the oviduct or uterus. However, during pregnancy or hormonal stimulation, transgene expression was markedly induced in the ovary, oviduct, and uterus. Furthermore, at placental implantation sites, high levels of transgene expression were observed. These data are consistent with previous reports on the regional expression of proenkephalin within the female reproductive system (10, 12-l 4, 20-22). In all three founder lines, animals had similar patterns of P-Gal expression in the reproductive system. Since the integration site of the transgene into the genome is random, it can be concluded that the integration site

did not strongly influence tissue-specific expression within the reproductive system. Therefore, the proenkephalin regulatory sequences contained within the construct appear to contain the necessary information for tissue-specific expression in both male and female reproductive systems. It is not yet clear which specific elements within the construct are essential. A construct with a shorter stretch of the human proenkephalin 5’flanking region, which we have previously analyzed in tissue culture models (2-5), has recently been shown to confer expression in the testis in three founder lines (23). However, no data are yet available for this shorter construct on the cell type-specific expression in the testis or the epididymis. It can be concluded that the human proenkephalin sequences contained within pENKP-Gal 2.3 target expression to appropriate cell types in both the male and female reproductive system and, in females, confer responsiveness to exogenous hormones and pregnancy. These results are of particular interest because of differences in the human and rodent proenkephalin genes. Kilpatrick et a/. (25, 38) have shown, for the rat and the mouse proenkephalin gene, that two species of proenkephalin RNA are found in somatic and germ cells. A 1450-nucleotide proenkephalin mRNA is present in the somatic cells (brain and testis), and a 1700nucleotide proenkephalin mRNA is the major form in mouse and rat spermatogenic cells (38). For the rat and

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Human

Prmnkephalin

Gene

1509

Expression

Fig. 7. Photographs

of Whole Mounts of Ovary &duct, and Uterus from Wild Type (A), Transgenic ENK Pregnant (C). and After Hormonal Manlpulatlon with PMS and hCG (Dj See Akterrals and Methods for details o, Ovary d. oviduct, u, uterus, the arrow rndlcates &Gal stalnlng around a developing oocyte (see Fig. 80 for more detail)

mouse proenkephalin gene, somatic and germ-cell promoters have been reported to be distinct (25). It has been shown that the major testicular proenkephalin mRNA species is transcribed from a promoter within the first intron of the gene. In contrast to the rat and mouse, the human (6) and hamster (10) proenkephalin mRNA does not contain the sequences corresponding to the testis-specific rat promoter. The construct introduced into the transgenic lines contains the human

promoter promoter.

and therefore

lacks the putative testis-specific

Nonetheless,

it still directs expression in It can be concluded that the mouse testis has all of the necessary transcriptional apparatus required to recognize the upstream promoter of the human proenkephalin gene.

spermatogenic

MATERIALS Production

cells.

AND of fransgemc

METHODS Animals

The DNA for injection into the mouse pronuclei was produced by a #otISphI digestion which removed the vector sequences from the ENK 2.3~$-Gal plasmid. The method of producing the transgenlc animals has been previously described (40). Briefly, 1 pl DNA solution (IO rig/ml) was injected into the pronucleus of fertilized one-cell eggs from superovulated (6602) Fl females mated by BGSJL Fl males (Jackson Laboratories: Bar Harbor, ME)~ These were then transferred into the oviduct of recipient pseudopregnant foster CD1 females (Charles River, Wilmington, MA). Positive animals. as determined by polym-

1 1 Nonpregnant

(B).

In the granulosa

cells

erase chain reactlon of tall ONA were bred to homwygosrty Founder lines (ENK 1 1. ENK 1 2. and ENK 1 3) were produced using this DNA sequence. Southern blots of the DNA-posltlve animals revealed a single integration site in each founder X-Gal

Staining

The chromogenic substrate X-Gal was used to detect expression of J-Gal produced by the transgene The method for XGal stalnlng was adapted from Price et al. (41). Mice were anesthetized with ip inlections of 0 4 ml 2.5% avertIn and then perfused with 3% formaldehyde In piperazine-N,N’-bis(2-ethanesulfonlc aad) (Sigma, St LOUIS, MO) solution, the tissue of Interest dissected out and placed in 30°10 sucrose overntght Frozen serial sectlons (45 microns) of tissue were placed on gelatin-coated slides Whole organs or slides were incubated In X-Gal solution overnight at 37 C The X-Gal (Jersey Laboratory Supply, New Jersey) dissolved in dimethylformamide (40 mgjml) was added to a mixer solutron to give a flnal concentration of 1 mg/ml The mixer solution was made up in PBS containing 35 mM K,Fe(CN),. 35 mM K4Fe(CQ ‘H,O. 2 rn~ MgCI,, 0 01% sodium deoxycholate, and 0.02°0 NP40 Slides were dehydrated In ascending concentrations of alcohol, dipped in xylene and cover slipped with mounting medium (Cytoseal, Stephens Scientific. New Jersey) Wild type C57BL\6J animals (Jackson Laboratories) were placed through the same protocol. Cell Isolation

of Spermatogenic

Cells

Germ cells were dissociated using a collagenase and trypsin treatment (42) and then fractionated on BSA gradients as previously described (43) Leydrg cells were Isolated folIowIng a similar collagenase treatment and gradient purification (44. 45) Cell purity was assessed by phase contrast microscopy

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MOL 1510

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Fig. 8. Secttons from the Ovary Stalned for $Gal Showing Low-Power (A and C) and High-Power (6 and D) Phase Contrast Photomlcrographs from Nonpregnant (A and B) and Pregnant (C and D) Mice The photographs Show high levels of :J-Gal expresslon In the granulosa cells surrounding the developing oocytes in panels C and D. Hormonal manipulation produced high levels of $Gal expresslon in a distribulion similar to that seen in pregnant animals Arrows point to developing oocytes.

Experimental

Manipulation

of Mice

Juvenile (P25) transgenic females ENK 1.1 and wild type animals injected ip with 50 IU hCG (Sigma) 48 h after receiving 5 IU PMS (Sigma) were examined. The animals were killed 5 h after the hCG injection and the tissue prepared as previously described (see X-Gal Stain&g).

Tissue derived from five adult males was used for RNA analysis of Isolated qerm cells (spermatocytes, round spermatlds, and elongating spermatlds) or Leydlg cells (see isolatlon and extraction methods above) After this. Northern analy SIS wab performed as described above for mRNA for endogenous proenkephalln and for LacZ mRNA (0.8 mg) was loaded In each lane

&Gal

In Situ

tmmunohistochemistry

Sectlons of adult mouse testis (20 pm) were thaw mounted onto gelatin-coated slides and fixed In 4% paraformaldehyde. The slides were Incubated for 48 h in +-Gal antiserum diluted 1 .lOOO lmmunodetectlon was performed using Vectistain ABC Elate Kit (Vector Laboratories. Burllngame CA) RNA

Preparation

and Northern

Blot Analysis

After rapid cervical dislocafion, the epididymis and testis were rapidly removed from animals. frozen on dry ice, and stored at -80 C until used. Total RNA was extracted by homogenization in guanidine thiocyanate according to the method of Chirgwin et al. (46). Total RNA from whole testis and epididymis was subjected to electrophoresis, transferred to a nylon membrane+ and hybridized with the indicated probes as described previously (47). “?nick translated hybridizations were not less that 5 x IO8 cpm/& DNA. After hybridization. the membranes were washed under stringent conditions including washes at 60 C in 0.2 standard s&ium citrate (SSC), 0.1% sodium dodecyl sulfate, and 0.1% sodium pyrophosphate. Autoradiography was performed with Kodak XAR-5 film (Eastman Kodak Co., Rochester, NY) at -70 C in the presence of an intensifying screen for l-l 2 h.

Hybridization

Adult mouse testes were rapldly dlssected out and Immersed in isopentane (-40 C for IO set The tissue was cut on a Jung cryostat (ReichertJung. Vienna, Austria) at 20 pm and thaw mounted onto gelatin-coated slides, postfixed with 4”0 paraformaldehyde in lx PBS for 5 min. After rinsing in PBS. the tissue was acetylated and placed rn graded ethanol A 1017. bp rlboprobe for rat proenkephalln was prepared using T3 or T7 polymerase reaction and the probe labeled with [3”S]dCTP . Thirty microliters of solution/section were used at 6X lo5 cpm/ lll. Hvbridiration was Derformed at 55 C in 50% formamide and ix SSC in 50 rnM phosphate buffer, 10 mM dithiothreitol. Denhardt s solution, 0 1 mg/ml yeast tRNA, and 0 1 mg/ml salmon sperm DNA After hybndlzatlon sections were treated with RNase A for 30 mln at 37 C and washed IIT SSC Sections were then dipped in Kodak NTB emulsion and kept for IO days at 4 C before development Sections were counterstained with 0.4% toluidine blue

Acknowledgments Received May 8, 1992 Accepted June 18 1992.

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Human

Proenkephalin

Gene

Expression

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Fig. 9. Whole Mount

Photographs of Uteri from Transgenic Animals Showing P-Gal Expression in the Uterine Wall of on the 7th and 12th days of Pregnancy (Panels A and B, Respectrvely) Sections through the uteroplacental junctron in a 7-day pregnant transgenic mouse shows B-Gal stainrng at the uteroplacental junction (C); arrows indicate the placental surface. For comparison a similar section (D) is stained wrth neutral red showing the uterus (u), the utero-placental junction (arrows) and the developing embryo (f). *, Uteroplacental junction in panels A and B.

Address requests for reprints to: Dr. David Borsook, Molecular Neurobiology Laboratory, Neuroscience Center, Massachusetts General Hospital, CNY 6, Building 149, 13th Street, Charlestown, Massachusetts 02129. Portions of this work were supported by NIH Grants MH44160 and MH-00892 (to S.E.H.) DA-05706 (to M.C.), NS18381, and NS-20591, a grant from the March of Dimes Birth Defects Foundation (to K.H.), and Dr. Richard J. Kitz, Department of Anesthesia, Massachusetts General Hospital (D.B.). * Current address: Alzheimer Research Laboratory, Case Western Reserve Medical School, 2116 Abington Road, Cleveland, Ohio 44106.

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