Local Regulation within the Female Reproductive System and upon Embryonic Implantation: Identification of Cells Expressing Proenkephalin A
Haim Rosen, Ahuva Itin, Rachel Schiff, and Eli Keshet Departments of Molecular Virology (H.R.) and Virology (A.I., R.S., E.K.) The Hebrew University Hadassah Medical School Jerusalem, Israel
The detection of proenkephalin A (PEA) mRNA and encoded peptides in various regions of the female reproductive system raised the possibility that opioid peptides might act as local regulators within this system. Assignment of a specific role for locally synthesized enkephalins has been hampered, however, by the unknown identity of the cells that produce PEA. Using in situ hybridization analysis we have now identified the cell types that express PEA mRNA in the reproductive system of female mice. In the ovary, PEA mRNA was localized primarily in theca cells of preovulatory follicles, and to a lesser extent, in follicular granulosa cells. In the oviducts, where PEA mRNA is most abundant, expression was confined to the secretory and ciliated epithelium of the mucosa. In the uterus, the site of PEA mRNA expression was the deep glandular layer of the endometrium. When pregnancy ensues, and upon decidual transformation, PEA expression by the same uterine cells was dramatically elevated. Elevated levels of PEA mRNA were detected predominantly in the vicinity of the implantation site, suggesting that signaling by the implanted embryo play a role in stimulating PEA expression. Based on these results, possible physiological roles for PEA-encoded peptides as autocrine/paracrine regulators within the female reproductive system are suggested. (Molecular Endocrinology 4:146-154,1990)
pituitary-gonadal axis through neuroendocrine mechanisms. The contention that local regulators also operate within the reproductive system is gaining increasing support. Local regulators are produced by distinct cells of the gonad and other parts of the reproductive tract. They are assumed to exert their physiological roles on receptor-bearing target cells within the reproductive system, presumably, in an autocrine or a paracrine manner. A variety of molecules have been implicated as local regulators of the female reproductive system. The list of putative local regulators of ovarian function includes oocyte maturation and gonadotrophin inhibitors, eicosanoids, GnRH-like peptides, growth factors, neurotransmitters, and neuropeptides. The latter group also includes opioid peptides (For a recent review, see Ref. 1). Opioid peptides in mammals are proteolytically processed from three precursor proteins encoded, each, by a single copy gene: proenkephalin A (PEA), prodynorphin, and POMC (2). Messenger RNAs encoded by all three genes, as well as their respective encoded peptides, were detected in different regions of the reproductive systems of both sexes (3-9). Although these findings clearly establish that opioids are produced within the reproductive system, assignment of a specific function for locally synthesized opioids has not yet been established. The present study focuses on the expression of PEA in the murine female reproductive system with the aim of identifying the cell types in which PEA mRNA is expressed. PEA is the precursor of the opioid peptides Metenkephalin, Leu-enkephalin, Met-enkephalin-Arg-Phe, Met-enkephalin-Arg-Gly-Leu, and Met-enkephalin-ArgArg-Val (10-12). PEA is also the precursor of peptides with no demonstrable opioid activity like amidorphin 826 and synenkephalin (13, 14). PEA mRNA was detected in the ovary, oviduct, and the uterus of several mammalian species (3, 4, 7,15-17). PEA-derived peptides were also detected in the female reproductive system of mice, rats, hamsters, cattle, and primates (4,
INTRODUCTION
Regulation of reproduction involves complex interactions between diverse cell types within the gonads and the reproductive tracts of both sexes. This complex regulation is coordinated primarily at the hypothalamic0888-8809/90/0146-0154$02.00/0 Molecular Endocrinology Copyright © 1990 by The Endocrine Society
146
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 18 November 2015. at 23:19 For personal use only. No other uses without permission. . All rights reserved.
Cell Type Specificity of Proenkephalin mRNA
18, 19). Moreover, levels of PEA mRNA in the ovary and uterus were found to change in a cyclic manner during the estrous cycle, to be up-regulated by administration of gonadal steroids and to be elevated during pregnancy (15-17). While it is well established that enkephalins systemically affect the reproductive system by modulating the secretion of gonadotrophins through a control of the pulsatile release of LHRH (20, 21), the local actions of enkephalins, might be exerted through other mechanisms. Identifying the expressing cell types and their immediate contacts are essential for the evaluation of putative local circuits, particularly, if one considers that PEA might act directly on the producer cell (autocrine regulation) or on nearby target cells (paracrine regulation). In this study, we employed in situ hybridization analysis, and have identified the cell types that synthesize PEA mRNA in the ovary, oviducts, and uterus of nonpregnant cycling female mice, and during early stages of pregnancy.
RESULTS
In order to determine the cell types that synthesize PEA we chose to detect PEA mRNA at its site of synthesis by in situ hybridization. The search for PEA mRNA rather than PEA-derived peptides was chosen for two reasons: First, peptides which are the end products of a proteolytic processing of the PEA precursor protein might be rapidly secreted and transported to distal sites and thus, be missing or misrepresented in the producer cell. Alternatively, the final processing of peptides detectable by immunological methods might take place in cells other than the one producing PEA mRNA and protein. Second, mRNA analysis is a more general test for PEA expression due to the fact that a single species of PEA mRNA encodes all PEA-derived peptides potentially generated from the PEA protein. As a PEA specific probe we employed a 278 base pair long PEA sequence fully contained within the enkephalin-encoding exon of the murine gene (see Materials and Methods for the derivation and characterization of the PEA-specific probe). 35S-Labeled RNA in an anti-sense orientation was synthesized in vitro and was used as an hybridization probe. For in situ hybridization 10-MITI thick frozen sections were prepared from organs of Balb/c female mice, and were processed and hybridized in situ under conditions that favor formation of mRNA: anti-sense cRNA hybrids (see Materials and Methods for experimental protocols). PEA Expressing Cells within the Ovary Previous studies have shown that steady-state levels of PEA mRNA in the ovaries of rats and hamsters changed markedly with the stage of the cycle, with maximal levels occurring between proestrus and estrus and with a rapid decline after metestrus (15). In order
147
to determine whether mice also exhibit a similar pattern of expression, and in order to determine more precisely the stage of maximal PEA expression (the stage to be selected for the in situ hybridization analysis) we performed a Northern blot analysis on RNAs extracted from PMSG-human CG (hCG) primed animals. Ovaries were hyperstimulated by successive injections of PMSG and hCG as described in Materials and Methods. As can be seen in Fig. 1 maximal levels of PEA mRNA were detected 5-10 h after administration of hCG. Densitometric tracings indicated a 10-fold stimulation relative to levels observed before the surge of hCG. The major events occurring in the ovary during that period are the germinal vesicle breakdown, completion of the first meiotic division, and preparation for ovulation (ovulation occurs 11-14 h after hCG injection). This period is also distinguished by a massive steroidogenic activity of theca and granulosa cells. The detection of a single 1.4 kilobase band by the PEA-specific probe and its identity to brain PEA mRNA also provided us with a further assurance to the validity of using this probe in the following in situ hybridization experiments. Animals treated with PMSG and hCG were killed and
origin —
JEJP mm 28 S — 18S — Proenk— \ m-mm-
- -
1.4 kb 1 2 Actin
3 4
5
6
Haim Rosen, Ahuva Itin, Rachel Schiff, and Eli Keshet Departments of Molecular Virology (H.R.) and Virology (A.I., R.S., E.K.) The Hebrew University Hadassah Medical School Jerusalem, Israel
The detection of proenkephalin A (PEA) mRNA and encoded peptides in various regions of the female reproductive system raised the possibility that opioid peptides might act as local regulators within this system. Assignment of a specific role for locally synthesized enkephalins has been hampered, however, by the unknown identity of the cells that produce PEA. Using in situ hybridization analysis we have now identified the cell types that express PEA mRNA in the reproductive system of female mice. In the ovary, PEA mRNA was localized primarily in theca cells of preovulatory follicles, and to a lesser extent, in follicular granulosa cells. In the oviducts, where PEA mRNA is most abundant, expression was confined to the secretory and ciliated epithelium of the mucosa. In the uterus, the site of PEA mRNA expression was the deep glandular layer of the endometrium. When pregnancy ensues, and upon decidual transformation, PEA expression by the same uterine cells was dramatically elevated. Elevated levels of PEA mRNA were detected predominantly in the vicinity of the implantation site, suggesting that signaling by the implanted embryo play a role in stimulating PEA expression. Based on these results, possible physiological roles for PEA-encoded peptides as autocrine/paracrine regulators within the female reproductive system are suggested. (Molecular Endocrinology 4:146-154,1990)
pituitary-gonadal axis through neuroendocrine mechanisms. The contention that local regulators also operate within the reproductive system is gaining increasing support. Local regulators are produced by distinct cells of the gonad and other parts of the reproductive tract. They are assumed to exert their physiological roles on receptor-bearing target cells within the reproductive system, presumably, in an autocrine or a paracrine manner. A variety of molecules have been implicated as local regulators of the female reproductive system. The list of putative local regulators of ovarian function includes oocyte maturation and gonadotrophin inhibitors, eicosanoids, GnRH-like peptides, growth factors, neurotransmitters, and neuropeptides. The latter group also includes opioid peptides (For a recent review, see Ref. 1). Opioid peptides in mammals are proteolytically processed from three precursor proteins encoded, each, by a single copy gene: proenkephalin A (PEA), prodynorphin, and POMC (2). Messenger RNAs encoded by all three genes, as well as their respective encoded peptides, were detected in different regions of the reproductive systems of both sexes (3-9). Although these findings clearly establish that opioids are produced within the reproductive system, assignment of a specific function for locally synthesized opioids has not yet been established. The present study focuses on the expression of PEA in the murine female reproductive system with the aim of identifying the cell types in which PEA mRNA is expressed. PEA is the precursor of the opioid peptides Metenkephalin, Leu-enkephalin, Met-enkephalin-Arg-Phe, Met-enkephalin-Arg-Gly-Leu, and Met-enkephalin-ArgArg-Val (10-12). PEA is also the precursor of peptides with no demonstrable opioid activity like amidorphin 826 and synenkephalin (13, 14). PEA mRNA was detected in the ovary, oviduct, and the uterus of several mammalian species (3, 4, 7,15-17). PEA-derived peptides were also detected in the female reproductive system of mice, rats, hamsters, cattle, and primates (4,
INTRODUCTION
Regulation of reproduction involves complex interactions between diverse cell types within the gonads and the reproductive tracts of both sexes. This complex regulation is coordinated primarily at the hypothalamic0888-8809/90/0146-0154$02.00/0 Molecular Endocrinology Copyright © 1990 by The Endocrine Society
146
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 18 November 2015. at 23:19 For personal use only. No other uses without permission. . All rights reserved.
Cell Type Specificity of Proenkephalin mRNA
18, 19). Moreover, levels of PEA mRNA in the ovary and uterus were found to change in a cyclic manner during the estrous cycle, to be up-regulated by administration of gonadal steroids and to be elevated during pregnancy (15-17). While it is well established that enkephalins systemically affect the reproductive system by modulating the secretion of gonadotrophins through a control of the pulsatile release of LHRH (20, 21), the local actions of enkephalins, might be exerted through other mechanisms. Identifying the expressing cell types and their immediate contacts are essential for the evaluation of putative local circuits, particularly, if one considers that PEA might act directly on the producer cell (autocrine regulation) or on nearby target cells (paracrine regulation). In this study, we employed in situ hybridization analysis, and have identified the cell types that synthesize PEA mRNA in the ovary, oviducts, and uterus of nonpregnant cycling female mice, and during early stages of pregnancy.
RESULTS
In order to determine the cell types that synthesize PEA we chose to detect PEA mRNA at its site of synthesis by in situ hybridization. The search for PEA mRNA rather than PEA-derived peptides was chosen for two reasons: First, peptides which are the end products of a proteolytic processing of the PEA precursor protein might be rapidly secreted and transported to distal sites and thus, be missing or misrepresented in the producer cell. Alternatively, the final processing of peptides detectable by immunological methods might take place in cells other than the one producing PEA mRNA and protein. Second, mRNA analysis is a more general test for PEA expression due to the fact that a single species of PEA mRNA encodes all PEA-derived peptides potentially generated from the PEA protein. As a PEA specific probe we employed a 278 base pair long PEA sequence fully contained within the enkephalin-encoding exon of the murine gene (see Materials and Methods for the derivation and characterization of the PEA-specific probe). 35S-Labeled RNA in an anti-sense orientation was synthesized in vitro and was used as an hybridization probe. For in situ hybridization 10-MITI thick frozen sections were prepared from organs of Balb/c female mice, and were processed and hybridized in situ under conditions that favor formation of mRNA: anti-sense cRNA hybrids (see Materials and Methods for experimental protocols). PEA Expressing Cells within the Ovary Previous studies have shown that steady-state levels of PEA mRNA in the ovaries of rats and hamsters changed markedly with the stage of the cycle, with maximal levels occurring between proestrus and estrus and with a rapid decline after metestrus (15). In order
147
to determine whether mice also exhibit a similar pattern of expression, and in order to determine more precisely the stage of maximal PEA expression (the stage to be selected for the in situ hybridization analysis) we performed a Northern blot analysis on RNAs extracted from PMSG-human CG (hCG) primed animals. Ovaries were hyperstimulated by successive injections of PMSG and hCG as described in Materials and Methods. As can be seen in Fig. 1 maximal levels of PEA mRNA were detected 5-10 h after administration of hCG. Densitometric tracings indicated a 10-fold stimulation relative to levels observed before the surge of hCG. The major events occurring in the ovary during that period are the germinal vesicle breakdown, completion of the first meiotic division, and preparation for ovulation (ovulation occurs 11-14 h after hCG injection). This period is also distinguished by a massive steroidogenic activity of theca and granulosa cells. The detection of a single 1.4 kilobase band by the PEA-specific probe and its identity to brain PEA mRNA also provided us with a further assurance to the validity of using this probe in the following in situ hybridization experiments. Animals treated with PMSG and hCG were killed and
origin —
JEJP mm 28 S — 18S — Proenk— \ m-mm-
- -
1.4 kb 1 2 Actin
3 4
5
6
