F’mstaglandins Leukotrienes and Essential 0 Longman Group UK Ltd 1992
Fatty Acids (1992) 47. 247-252
Immunohistochemical Localization of PGF,, Receptor in the Mouse Testis D. J. Orlicky and C. Williams-Skipp Departments of Pathology, University of Colorado Health Sciences Center, and University of Colorado Cancer Center, 4200 East Ninth Avenue, Denver, CO 80262, USA (Reprint requests to DJO) ABSTRACT. As a step towards understanding the role of prostaglandin F, (PGF& in male reproductive tract
physiology, a rabbit polyclonal antiserum reactive with purified PGF2, receptor (PGF,,-R) was produced. Here we describe the use of this anti-PGF,,-R antiserum in immunohistochemical staining of mouse testis to ascertain which cell types, in vivo, possess immunoreactive PGF%,-R.As an initial control Western blot analysis was performed to show that the anti-PGF,,-R antiserum recognizes only one antigen in the testis, and that this molecule is similar in molecular mass (by PAGE) to the previously described, purified PGF,,-R molecule. Immunohistochemical staining demonstrates that adult mouse testis contains a single subpopulation of cells with PGF,-R and that subpopulation is the interstitial or Leydig cell subpopulation. Cell and tissue types negative for immunoreactive PGF&-R include: the capsule (tunica albuginea) and subcapsular stroma, all histologic layers of the vasculature (both venules and arterioles), peritubular stroma, peritubular boundary tissue, spermatogonia, primary and secondary spermatocytes, spermatids, Sertoli cells, and spermatozoa. While the above described localization of PGF2,-R is also seen in rat, there are fewer rat Leydig cells and this subpopulation appears to atrophy and stain less intensely with increasing age of the animal. Preabsorption of the PGF,,-R antiserum with a corpora lutea homogenate acetone powder eliminated immunohistochemical staining of the Leydig cell subpopulation further suggesting that the antigenic determinant detected here is related to that in the ovary (PGF,,-R).
INTRODUCTION
sterone synthesis (12, 13). Not all investigators have attributed the PGF*, effect on Leydig cell testosterone synthesis to a direct PGF,,-Leydig cell interaction. These investigators (4, 5) suggest that the decrease in testosterone synthesis is an indirect effect resulting from a PGF*,-induced decrease in testicular blood flow. It should be noted that other investigators have observed no effect of PGF,, on testosterone synthesis (14). Our approach toward understanding how PGF,, works in vivo is to first identify which tissues are affected directly by PGF,,, i.e. to assess which tissues are able to bind [3H]PGF,,. Next, responsive tissues are examined by immunohistochemistry to determine precisely which cells actually express the PGF*, receptor. To accomplish the latter, we have purified the PGF*, receptor (PGF,,-R) from bovine corpora lutea and produced a rabbit polyclonal antiserum against it for use in immunohistochemical studies ( 15, 16).
It has been suggested that prostaglandin (PG)F2, may mediate a number of processes important in normal male reproductive tract physiology. These processes can be divided into those involving and not involving the testis. Non-testicular processes include: a) growth promoting effects on rat seminal vesicle and ventral prostate (l), b) an increase in human spermatozoa motility (2) or an inhibition of human sperm motility (3), and c) unknown effects on human prostate tissue (PGF2, is thought to affect prostatic tissue due to its ability to bind to this tissue) (6). Testicular responses to PGF,, include: a) inhibition of testicular blood flow (rat; 4, 5), and b) an effect on testosterone synthesis (8-13). With regard to the latter process, [3H]PGF,, has been shown to bind to rat Leydig cells (7). Furthermore, PGF,, can either increase bull or rhesus monkey Leydig cell synthesis of testosterone (8,9) or decrease rat and mouse Leydig cell testosterone (10-l 3) synthesis. PGF,, is also reported to inhibit the luteinizing hormone stimulation of rat testo-
MATERIALS AND METHODS Immunohistochemistry ICR mice (2-4 months old) were obtained from Harlan Sprague Dawley Inc, Indianapolis, IN. Animals were
Date received 23 June 1992 Date accepted 16 July 1992 241
248
Prostaglandins
Leukotrienes
and Essential Fatty Acids
killed by cervical dislocation. Tissues were immediately excised and fixed in Bouin’s fixative with agitation, for 4 h at room temperature. They were then routinely paraffin processed. Sections (5 p) were cut and stained by the peroxidase-antiperoxidase method of Stemberger (17) using a 1:250 dilution of preimmune or immune sera (15), 1:80 dilution of swine antirabbit immunoglobulins (Dako Corporation, Carpinteria, CA) and a 1:220 dilution of rabbit peroxidase-antiperoxidase conjugate (ICN Immunobiologicals, Lisle, IL). The staining was developed with diaminobenzidine and H202, then the section was lightly counterstained with hematoxylin. All antiserum, immune and preimmune, was preabsorbed with a liver acetone powder (10 mg/ml diluted antiserum) as previously described. Furthermore, all slides contained a small piece of rat ovary which acted as a control for specificity and intensity of staining (16). Young (30-day-old) and adult (?250 g) F344 (Fisher) rats were obtained from the Harlan Sprague Dawley Animal Facility (Fredrick, MD). Animals were killed by COZ asphyxiation followed by cervical dislocation. Tissues were excised, fixed, processed and stained as described above for mouse tissues. Western blot Whole tissue was excised from freshly killed animals, immediately homogenized and precipitated with acetone as previously described (15). Briefly, the protein content
of the samples was quantitated, the samples were separated by polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose, and the transferred protein was probed with the antiserum (1:250). All antiserum (preimmune, immune, link and PAP) was preabsorbed with a liver acetone powder (lOmg/ml diluted antiserum) and was used in the presence of 1% normal swine serum as done in immunohistochemical staining. Preblocking the nitrocellulose-protein blot with 1% normal swine serum also significantly reduced the background staining.
RESULTS Figure 1 shows a photograph of a Western blot performed using immune and preimmune anti-PGF,,-R antiserum. The starting material in lane A and B was rat corpora lutea homogenate and in lane C and D whole mouse testis homogenate (80 pg protein per lane). Following staining with immune antiserum (lanes A and C), a positive band is seen corresponding to a molecular weight of approximately 133 kD. This molecular weight is similar to that seen in PGF,,-R of bovine corpora lutea origin (135 kD) and is the same as we have previously reported for PGF,,-R of rat corpora lutea origin (15). Furthermore, the presence of but a single band suggests that, at least under these conditions and using these tissues, this antibody is specific for the PGF*,-R protein.
Fig. 1 Western blot analysis of PGF,,-R. Rat corpora lutea homogenate (lane A, B) and mouse testis homogenate (lane C. D) was prepared, acetone precipitated, separated by 6.25% PAGE, transferred to nitrocellulose paper, and probed with either immune antiserum (A, C) or preimmune antiserum (B, D) as described in Materials and Methods. Relative position of molecular weight markers, bromphenol blue dye front (df) and origin (ori) are indicated.
Immunohistochemical Localization of PGFla Receptor in the Mouse Testis
No staining is seen following use of the preimmune serum (lanes B and D). The photomicrographs in Figure 2 present results concerning four aspects of PGF*,-R immunohistochemical localization in the testes. These include localization in a) mouse tissue, b) young and old rat tissue, c) vasculature and d) the immunological crossreactivity of testis PGF,,-R with ovary PGF,,-R. First of all PGF2,-R staining is shown in mouse testis (Fig. 2. A-F). A hematoxylin and eosin stained section (A) is included to show the quality of the starting material and the relatively large number of interstitial cells in mouse testis. Figure 2B and 2C show immune anti-PGF,,-R antiserum staining of mouse testis at low (B) and high (C) magnification. Interstitial cells alone, among all the cell types in the testis, stain for the presence of PGF,,-R. The staining is heterogeneous and it would appear that some interstitial cells only stain very weakly, if at all. Preimmune staining of mouse testis is also seen at low (E) and high (F) magnifications. No cells in the testis stain positive when stained with preimmune antiserum. A higher concentration (1:200) of immune antiserum was also tried in the staining reaction in an attempt to increase the sensitivity of the assay; however, this concentration only increased the diffuse background staining without showing specific PGF?,-R staining of any additional cell types. Second, PGF,,-R staining is shown in rat testis of young (30-day-old) and adult (>250 g) animals (Fig. 2H and 21, respectively). A hematoxylin and eosin stained section (2G) of young, but not adult, testis is included to show the quality of the starting material and to show the relative paucity of interstitial cells in rat testis as compared to mouse testis. The adult testis was of similar quality and had approximately an equal or slightly lower number of interstitial cells as the young testis (data not shown). PGF,,-R immunohistochemical staining of young testis revealed a small number of intensely staining and some weakly positive or non-staining interstitial cells. On the other hand, PGF2,-R staining of adult testis indicated that most interstitial cells were either weakly positive or non-staining. No intensely staining cells were seen in adult testis. Preimmune staining of rat testis was as negative as for mouse testis and, therefore, is not shown. Third. PGFz,-R staining is shown in both mouse (Fig. 2J) and rat (Fig. 2K. 2L) testicular vasculature. In none of these micrographs are vessels seen to stain for the presence of PGF,,-R. however, in all cases immunopositive interstitial cells are seen in close juxtaposition to the vessels. Fourth, PGF,,-R immunohistochemical staining of mouse testis is shown following preabsorption of the immune antiserum with an ovary acetone powder (Fig. 2D). The amount of this ovary acetone powder (10 mg/ml) is the same as the amount of liver acetone powder which all immune and preimmune antiserum has been preabsorbed with. The staining seen in this phot-
249
omicrograph is very light compared with Figure 2C suggesting some component of the rat ovary shows a similar antigenic determinant to that of the mouse testis. By increasing the amount of rat ovary acetone powder 5fold it was possible to completely inhibit all subsequent immunohistochemical staining of mouse testis (data not shown). Summarizing the findings in Figure 2 it is seen that only a single testicular subpopulation of cells contain immunoreactive PGF,,-R, and that subpopulation is the interstitial or Leydig cell subpopulation. Also apparent are a number of cell and tissue types that do not contain immunoreactive PGF,,-R. Immunohistochemically negative tissues include: the capsule or tunica albuginea, the subcapsular stroma, peritubular stroma, all the histologic layers of the testicular vasculature (both venules and arterioles), peritubular boundary tissue, spermatogonia, primary and secondary spermatocytes. spermatids, Sertoli cells and spermatozoa. Rat testicular cell subpopulations stain qualitatively the same as in the mouse, however. quantitatively less intensely. A subset of the young rat Leydig cells seem to stain brighter than adult Leydig cells and approximately as bright as mouse Leydig cells.
DISCUSSION Prostaglandins were first described in secretions of male reproductive tract origin and since then have been variously ascribed roles in both the male and female reproductive tracts. Many studies have described synthesis of these hormones by either whole in vivo or separated in vitro tissues allowing for accurate determination of the sources and stimuli for synthesis. However, assessing which tissues and which ceils in those tissues respond to these hormones directly (as opposed to indirectly - that is responding to mediators synthesized in response to prostaglandin stimulation) has been a good deal more difficult. Our approach to this problem has been to first purify the PGF,, receptor and then to assess which tissues possess this molecule. Here we describe our initial studies on localization of the PGF,,-R in the male reproductive tract. The anti-PGF?,-R antiserum used in these studies recognizes a protein antigen in mouse and rat tissues of similar size to that described in bovine corpora lutea (15). This antiserum has previously been used to localize PGF,,-R containing cells in the rat ovary (16). Interestingly, the ovary cells that contain this receptor were all cells thought to synthesize sex steroid hormones. An extension of this hypothesis was the possibility that cells that synthesize male sex steroid hormone also possess and respond to stimulation of the PGF,,-R. A number of investigators (7-14) have previously examined the interaction and role of PGFZ, in the testis. Most of these reports (7-13) have suggested an effect of PGF,, on testosterone synthesis although what that effect was and
250 Prostaglandins Leukotrienes and Essential Fatty Acids
Immunohistochemical
how it was produced was not agreed on. Apparently the species of the experimental animal is quite important. Both the bull and rhesus monkey increase testosterone synthesis in response to PGFza (8, 9) whereas both the mouse and rat decrease testosterone synthesis in response to PGF1, (10-13). The mechanism of this PGF,, response may be either a direct effect on Leydig cells (10-13) or an indirect effect resulting from a PGF,,-induced decrease in testicular blood flow (4, 5). Pertinent to this question, our present results clearly demonstrate the presence of PGF,,-R on Leydig cells and a lack of PGF2,-R on any vascular cell in vessels found in the testis. The possibility does exist, however, that a noncrossreactive PGF2,-R could be present on vascular tissue. Our present experiments directed at making a monoclonal antibody capable of blocking the binding of PGF,, to its receptor should help clarify this issue. Nonetheless, our data favor a direct effect of PGF,, on Leydig cell testosterone synthesis and an indirect effect of PGF2, on vasoconstriction in the mouse and rat testis. Certainly, we do observe PGF,,-R positive interstitial cells in close proximity to testicular vasculature which would facilitate an indirect mechanism of action. One other possibility that might yield immunonegative appearing vascular cells would be the presence of only a very low number of PGF2,-R on these cells. It is unknown at present what the sensitivity of this immunohistochemical technique is. The presence of PGF2,-R on Leydig cells and their role in inhibition of testosterone is of considerable interest in light of our previous findings that estrogen can up-regulate the PGF,,-R ( 18). For a number of years it has been known that estrogen stimulation of the male decreases testosterone synthesis (19-21), and more recently estrogen receptors have been demonstrated in the testis on Leydig cells (22-24). These previous findings, in view of ours, lead to the hypothesis that estrogen can regulate the synthesis of testosterone through PGF,, and the PGF?,-R. If this mechanism is correct, the combined actions of estrogen and PGF2, may be important during normal physiology or conceivably could be an important means to control testosterone synthesis in those clinical cases where this is of benefit. Lastly, it is important to note the magnitude of difference in quantity of Leydig cells and receptors on those Leydig cells of mice and rats. Previously, other investigators have noted the difference in number of Leydig cells between different species (reviewed in 25, 26) and an age-related decrease in the number of Leydig cells and the testosterone synthesis by those Leydig cells (25, 27). Here, we have also observed a decrease in intensity of PGF,,-R staining with aging. In consideration of all these data, the mouse may be a more appropriate animal model system to study in future experiments due to the larger number of Leydig cells, the larger amounts of immunohistochemically detectable PGF*,-R and the higher levels of testosterone synthesis in mouse testis. In conclusion, we have initiated experiments on male
Localization
of PGFr, Receptor in the Mouse Testis
251
reproductive tract tissue with the aim of determining which tissues can respond directly to PGF2,. Our present approach involves the immunohistochemical localization of PGF2, receptors. In the testis, these receptors appear to be confined to Leydig cells. We hypothesize that this may be important in the normal physiology of male sex steroids and that it may potentially be useful in certain clinical situations.
Acknowledgements This work was supported by NIH Grant HD25961-01 to DJO. We also thank N. Hart and C. Mraz for their expert secretarial assistance, and Drs P. Shanley and G. Miller for their critical review of this manuscript.
References 1. Chinoy N J, Sharma J D, Seethalakshmi L, Sanjeevan A G. Effects of prostaglandins on histology of male reproductive organs and fertility in rats. Int J of Fertil 1980; 25: 267-214 2. Eskin B A, Azarbal S, Sepic R, Slate W G. In vitro responses of the spermatozoa-cervical mucus system treated with prostaglandin Fra. Obstet Gynecol 1973; 41: 436-139 3. Cohen M S. Colin M J, Golimbu M. Hot&kiss R S. The effects of prostaglandins on sperm motility. Fertil Stern 1977; 28: 78-85 4. Free M J. Jaffe R A. Effects of prostaglandins on blood flow and pressure in the conscious rat. Prostaglandins 1972: 1: 483-498 5. Einer-Jensen N, Soofi G. Decreased blood flow through rat testis after intratesticular injection of PGFza Prostaglandins 1974 7: 377-382 6. Cavanaugh A H, Farnsworth WE. Receptor sites on human prostate tissue for prostaglandin FZ alpha. Life Sci 1977; 21: 83-92 7. Haour F. Kouznetzova B, Dray F, Saez J M. hCGinduced prostaglandin EZ and Fza release in adult rat testis: role in Leydig cell desensitization to hCG. Life Sci 1979: 24: 2151- 2158 8. Haynes N B. Hafs H D, Waters R J. Manns J G, Riley A. Stimulatory effect of prostaglandin F,, on the plasma concentration of testosterone in bulls. J Endocrinol 1975: 66: 329-338, 9. Kimball F A, Kirton K T, Forbes A D, et al. Serum FSH, LH and testosterone in the male rhesus monkey following prostaglandin injection. Prostaglandins 1979: 18: 117-122 10. Bartke A, Mosto N, Caldwell B X, Behnnan H R. Effects of a cholesterol esterase inhibitor and of prostaglandin Fza on testis cholesterol and on plasma testosterone in mice. Prostaglandins 1973: 3: 97-104 11. Saksena S K, El Safoury S, Bartke A. Prostaglandins EZ and FZa decrease plasma testosterone levels in male rats. Prostaglandins 1973: 4: 235-242 12. Grotjan H E, Heindel J J. Steinberger E. Prostaglandin inhibition of testosterone production induced by luteinizing hormone. dibutyryl cyclic AMP or 3-isobutyll-methyl xanthine in dispersed rat testicular interstitial cells. Steroids 1978; 32: 307-322 13. Fuchs A-R, Chantharaksri U. Prostaglandin Fzu regulation of LH-stimulated testosterone production in rat testis. Biol Reprod 198 1: 25: 492-50 1 14. Yamada K, Ohkura N, Satoh T. Reduction of testosterone levels in rats by prostaglandin Ez, Res Communi Chem Path01 Pharmacol 1985: 47: 153-156 15. Orlicky D J, Miller G J, Evans R M. Identification and purification of a bovine corpora luteal membrane glycoprotein with [3H]prostaglandin Fra binding properties. Prostaglandins Leukotr Essent Fatty Acids 1990: 41: 5161
252
Prostaglandins
Leukotrienes
and Essential Fatty Acids
16. Orlicky D J, Fisher L, Dunscomb N, Miller G J. Immunohistochemical localization of PGF,, receptor in the rat ovary. Prostaglandins Leukotr Essent Fatty Acids 1992; 4633: 223-229 17. Stemberger L A. Hardy P H, Cuculis J J, Meyer H G. The unlabeled antibody enzyme method of immunohistochemistry. Preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-antihorseradish peroxidase) and its use in identification of spirochetes. J Histochem Cytochem 1970; 18: 315-333 18. Orlicky D J, Lieberman R, Williams C, Gerschenson L E. Requirement for prostaglandin Fz,, in 178estradiol stimulation of DNA synthesis in rabbit endometrial cultures. J Cell Physiol 1987; 130: 292-300 19. Albert A. The mammalian testis. In: Young W C, ed. Sex and internal secretions. WiHiams and Wilkins, Baltimore, MD, 1961: 305-365 20. Chowdhury M, Tcholakian R, Steinberger E. An unexpected effect of oestradiol-17pon luteinizing hormone and testosterone. J Endocrinol 1974; 60: 375-376
21. Bartke A, Williams K I H. Dalterio S. Effects of estrogens on testicular testosterone production in vitro. Biol Reprod 1977; 17: 645649 22. Abney T 0. A cytoplasmic estradiol receptor in rat testicular tissue Endocrinol 1976; 99: 555- 563 23. Lin T, Chen G C, Murono E P, Osterman J, Nankin H R. Estradiol receptors of two distinct populations of Leydig cells. Steroids 1982; 40: 53-63 24. Terakawa N, Aono T, Matsumoto K. Presence of two types of estrogen binding sites in mouse testis. J Steroid Biochem 1985; 22: 147-150 25. deKretser D M, Kerr J B. The cytology of the testis. In: Knobil E. Neil1 J D, eds. The physiology of reproduction. Raven Press. New York, 1988: 837-932 26. Setchell B P, Brooks D E. Anatomy, vasculature, innervation, and fluids of the male reproductive tract. In: Knobil E, Neil1 J D, eds. The physiology of reproduction. Raven Press, New York, 1988: 753-836 27. Miller A E, Reigle G D. Aging effects of hypothalamic catecolamine and testosterone secretion in the male rat. Fed Proc 1975: 34: 303 (Abstract #481)
Fatty Acids (1992) 47. 247-252
Immunohistochemical Localization of PGF,, Receptor in the Mouse Testis D. J. Orlicky and C. Williams-Skipp Departments of Pathology, University of Colorado Health Sciences Center, and University of Colorado Cancer Center, 4200 East Ninth Avenue, Denver, CO 80262, USA (Reprint requests to DJO) ABSTRACT. As a step towards understanding the role of prostaglandin F, (PGF& in male reproductive tract
physiology, a rabbit polyclonal antiserum reactive with purified PGF2, receptor (PGF,,-R) was produced. Here we describe the use of this anti-PGF,,-R antiserum in immunohistochemical staining of mouse testis to ascertain which cell types, in vivo, possess immunoreactive PGF%,-R.As an initial control Western blot analysis was performed to show that the anti-PGF,,-R antiserum recognizes only one antigen in the testis, and that this molecule is similar in molecular mass (by PAGE) to the previously described, purified PGF,,-R molecule. Immunohistochemical staining demonstrates that adult mouse testis contains a single subpopulation of cells with PGF,-R and that subpopulation is the interstitial or Leydig cell subpopulation. Cell and tissue types negative for immunoreactive PGF&-R include: the capsule (tunica albuginea) and subcapsular stroma, all histologic layers of the vasculature (both venules and arterioles), peritubular stroma, peritubular boundary tissue, spermatogonia, primary and secondary spermatocytes, spermatids, Sertoli cells, and spermatozoa. While the above described localization of PGF2,-R is also seen in rat, there are fewer rat Leydig cells and this subpopulation appears to atrophy and stain less intensely with increasing age of the animal. Preabsorption of the PGF,,-R antiserum with a corpora lutea homogenate acetone powder eliminated immunohistochemical staining of the Leydig cell subpopulation further suggesting that the antigenic determinant detected here is related to that in the ovary (PGF,,-R).
INTRODUCTION
sterone synthesis (12, 13). Not all investigators have attributed the PGF*, effect on Leydig cell testosterone synthesis to a direct PGF,,-Leydig cell interaction. These investigators (4, 5) suggest that the decrease in testosterone synthesis is an indirect effect resulting from a PGF*,-induced decrease in testicular blood flow. It should be noted that other investigators have observed no effect of PGF,, on testosterone synthesis (14). Our approach toward understanding how PGF,, works in vivo is to first identify which tissues are affected directly by PGF,,, i.e. to assess which tissues are able to bind [3H]PGF,,. Next, responsive tissues are examined by immunohistochemistry to determine precisely which cells actually express the PGF*, receptor. To accomplish the latter, we have purified the PGF*, receptor (PGF,,-R) from bovine corpora lutea and produced a rabbit polyclonal antiserum against it for use in immunohistochemical studies ( 15, 16).
It has been suggested that prostaglandin (PG)F2, may mediate a number of processes important in normal male reproductive tract physiology. These processes can be divided into those involving and not involving the testis. Non-testicular processes include: a) growth promoting effects on rat seminal vesicle and ventral prostate (l), b) an increase in human spermatozoa motility (2) or an inhibition of human sperm motility (3), and c) unknown effects on human prostate tissue (PGF2, is thought to affect prostatic tissue due to its ability to bind to this tissue) (6). Testicular responses to PGF,, include: a) inhibition of testicular blood flow (rat; 4, 5), and b) an effect on testosterone synthesis (8-13). With regard to the latter process, [3H]PGF,, has been shown to bind to rat Leydig cells (7). Furthermore, PGF,, can either increase bull or rhesus monkey Leydig cell synthesis of testosterone (8,9) or decrease rat and mouse Leydig cell testosterone (10-l 3) synthesis. PGF,, is also reported to inhibit the luteinizing hormone stimulation of rat testo-
MATERIALS AND METHODS Immunohistochemistry ICR mice (2-4 months old) were obtained from Harlan Sprague Dawley Inc, Indianapolis, IN. Animals were
Date received 23 June 1992 Date accepted 16 July 1992 241
248
Prostaglandins
Leukotrienes
and Essential Fatty Acids
killed by cervical dislocation. Tissues were immediately excised and fixed in Bouin’s fixative with agitation, for 4 h at room temperature. They were then routinely paraffin processed. Sections (5 p) were cut and stained by the peroxidase-antiperoxidase method of Stemberger (17) using a 1:250 dilution of preimmune or immune sera (15), 1:80 dilution of swine antirabbit immunoglobulins (Dako Corporation, Carpinteria, CA) and a 1:220 dilution of rabbit peroxidase-antiperoxidase conjugate (ICN Immunobiologicals, Lisle, IL). The staining was developed with diaminobenzidine and H202, then the section was lightly counterstained with hematoxylin. All antiserum, immune and preimmune, was preabsorbed with a liver acetone powder (10 mg/ml diluted antiserum) as previously described. Furthermore, all slides contained a small piece of rat ovary which acted as a control for specificity and intensity of staining (16). Young (30-day-old) and adult (?250 g) F344 (Fisher) rats were obtained from the Harlan Sprague Dawley Animal Facility (Fredrick, MD). Animals were killed by COZ asphyxiation followed by cervical dislocation. Tissues were excised, fixed, processed and stained as described above for mouse tissues. Western blot Whole tissue was excised from freshly killed animals, immediately homogenized and precipitated with acetone as previously described (15). Briefly, the protein content
of the samples was quantitated, the samples were separated by polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose, and the transferred protein was probed with the antiserum (1:250). All antiserum (preimmune, immune, link and PAP) was preabsorbed with a liver acetone powder (lOmg/ml diluted antiserum) and was used in the presence of 1% normal swine serum as done in immunohistochemical staining. Preblocking the nitrocellulose-protein blot with 1% normal swine serum also significantly reduced the background staining.
RESULTS Figure 1 shows a photograph of a Western blot performed using immune and preimmune anti-PGF,,-R antiserum. The starting material in lane A and B was rat corpora lutea homogenate and in lane C and D whole mouse testis homogenate (80 pg protein per lane). Following staining with immune antiserum (lanes A and C), a positive band is seen corresponding to a molecular weight of approximately 133 kD. This molecular weight is similar to that seen in PGF,,-R of bovine corpora lutea origin (135 kD) and is the same as we have previously reported for PGF,,-R of rat corpora lutea origin (15). Furthermore, the presence of but a single band suggests that, at least under these conditions and using these tissues, this antibody is specific for the PGF*,-R protein.
Fig. 1 Western blot analysis of PGF,,-R. Rat corpora lutea homogenate (lane A, B) and mouse testis homogenate (lane C. D) was prepared, acetone precipitated, separated by 6.25% PAGE, transferred to nitrocellulose paper, and probed with either immune antiserum (A, C) or preimmune antiserum (B, D) as described in Materials and Methods. Relative position of molecular weight markers, bromphenol blue dye front (df) and origin (ori) are indicated.
Immunohistochemical Localization of PGFla Receptor in the Mouse Testis
No staining is seen following use of the preimmune serum (lanes B and D). The photomicrographs in Figure 2 present results concerning four aspects of PGF*,-R immunohistochemical localization in the testes. These include localization in a) mouse tissue, b) young and old rat tissue, c) vasculature and d) the immunological crossreactivity of testis PGF,,-R with ovary PGF,,-R. First of all PGF2,-R staining is shown in mouse testis (Fig. 2. A-F). A hematoxylin and eosin stained section (A) is included to show the quality of the starting material and the relatively large number of interstitial cells in mouse testis. Figure 2B and 2C show immune anti-PGF,,-R antiserum staining of mouse testis at low (B) and high (C) magnification. Interstitial cells alone, among all the cell types in the testis, stain for the presence of PGF,,-R. The staining is heterogeneous and it would appear that some interstitial cells only stain very weakly, if at all. Preimmune staining of mouse testis is also seen at low (E) and high (F) magnifications. No cells in the testis stain positive when stained with preimmune antiserum. A higher concentration (1:200) of immune antiserum was also tried in the staining reaction in an attempt to increase the sensitivity of the assay; however, this concentration only increased the diffuse background staining without showing specific PGF?,-R staining of any additional cell types. Second, PGF,,-R staining is shown in rat testis of young (30-day-old) and adult (>250 g) animals (Fig. 2H and 21, respectively). A hematoxylin and eosin stained section (2G) of young, but not adult, testis is included to show the quality of the starting material and to show the relative paucity of interstitial cells in rat testis as compared to mouse testis. The adult testis was of similar quality and had approximately an equal or slightly lower number of interstitial cells as the young testis (data not shown). PGF,,-R immunohistochemical staining of young testis revealed a small number of intensely staining and some weakly positive or non-staining interstitial cells. On the other hand, PGF2,-R staining of adult testis indicated that most interstitial cells were either weakly positive or non-staining. No intensely staining cells were seen in adult testis. Preimmune staining of rat testis was as negative as for mouse testis and, therefore, is not shown. Third. PGFz,-R staining is shown in both mouse (Fig. 2J) and rat (Fig. 2K. 2L) testicular vasculature. In none of these micrographs are vessels seen to stain for the presence of PGF,,-R. however, in all cases immunopositive interstitial cells are seen in close juxtaposition to the vessels. Fourth, PGF,,-R immunohistochemical staining of mouse testis is shown following preabsorption of the immune antiserum with an ovary acetone powder (Fig. 2D). The amount of this ovary acetone powder (10 mg/ml) is the same as the amount of liver acetone powder which all immune and preimmune antiserum has been preabsorbed with. The staining seen in this phot-
249
omicrograph is very light compared with Figure 2C suggesting some component of the rat ovary shows a similar antigenic determinant to that of the mouse testis. By increasing the amount of rat ovary acetone powder 5fold it was possible to completely inhibit all subsequent immunohistochemical staining of mouse testis (data not shown). Summarizing the findings in Figure 2 it is seen that only a single testicular subpopulation of cells contain immunoreactive PGF,,-R, and that subpopulation is the interstitial or Leydig cell subpopulation. Also apparent are a number of cell and tissue types that do not contain immunoreactive PGF,,-R. Immunohistochemically negative tissues include: the capsule or tunica albuginea, the subcapsular stroma, peritubular stroma, all the histologic layers of the testicular vasculature (both venules and arterioles), peritubular boundary tissue, spermatogonia, primary and secondary spermatocytes. spermatids, Sertoli cells and spermatozoa. Rat testicular cell subpopulations stain qualitatively the same as in the mouse, however. quantitatively less intensely. A subset of the young rat Leydig cells seem to stain brighter than adult Leydig cells and approximately as bright as mouse Leydig cells.
DISCUSSION Prostaglandins were first described in secretions of male reproductive tract origin and since then have been variously ascribed roles in both the male and female reproductive tracts. Many studies have described synthesis of these hormones by either whole in vivo or separated in vitro tissues allowing for accurate determination of the sources and stimuli for synthesis. However, assessing which tissues and which ceils in those tissues respond to these hormones directly (as opposed to indirectly - that is responding to mediators synthesized in response to prostaglandin stimulation) has been a good deal more difficult. Our approach to this problem has been to first purify the PGF,, receptor and then to assess which tissues possess this molecule. Here we describe our initial studies on localization of the PGF,,-R in the male reproductive tract. The anti-PGF?,-R antiserum used in these studies recognizes a protein antigen in mouse and rat tissues of similar size to that described in bovine corpora lutea (15). This antiserum has previously been used to localize PGF,,-R containing cells in the rat ovary (16). Interestingly, the ovary cells that contain this receptor were all cells thought to synthesize sex steroid hormones. An extension of this hypothesis was the possibility that cells that synthesize male sex steroid hormone also possess and respond to stimulation of the PGF,,-R. A number of investigators (7-14) have previously examined the interaction and role of PGFZ, in the testis. Most of these reports (7-13) have suggested an effect of PGF,, on testosterone synthesis although what that effect was and
250 Prostaglandins Leukotrienes and Essential Fatty Acids
Immunohistochemical
how it was produced was not agreed on. Apparently the species of the experimental animal is quite important. Both the bull and rhesus monkey increase testosterone synthesis in response to PGFza (8, 9) whereas both the mouse and rat decrease testosterone synthesis in response to PGF1, (10-13). The mechanism of this PGF,, response may be either a direct effect on Leydig cells (10-13) or an indirect effect resulting from a PGF,,-induced decrease in testicular blood flow (4, 5). Pertinent to this question, our present results clearly demonstrate the presence of PGF,,-R on Leydig cells and a lack of PGF2,-R on any vascular cell in vessels found in the testis. The possibility does exist, however, that a noncrossreactive PGF2,-R could be present on vascular tissue. Our present experiments directed at making a monoclonal antibody capable of blocking the binding of PGF,, to its receptor should help clarify this issue. Nonetheless, our data favor a direct effect of PGF,, on Leydig cell testosterone synthesis and an indirect effect of PGF2, on vasoconstriction in the mouse and rat testis. Certainly, we do observe PGF,,-R positive interstitial cells in close proximity to testicular vasculature which would facilitate an indirect mechanism of action. One other possibility that might yield immunonegative appearing vascular cells would be the presence of only a very low number of PGF2,-R on these cells. It is unknown at present what the sensitivity of this immunohistochemical technique is. The presence of PGF2,-R on Leydig cells and their role in inhibition of testosterone is of considerable interest in light of our previous findings that estrogen can up-regulate the PGF,,-R ( 18). For a number of years it has been known that estrogen stimulation of the male decreases testosterone synthesis (19-21), and more recently estrogen receptors have been demonstrated in the testis on Leydig cells (22-24). These previous findings, in view of ours, lead to the hypothesis that estrogen can regulate the synthesis of testosterone through PGF,, and the PGF?,-R. If this mechanism is correct, the combined actions of estrogen and PGF2, may be important during normal physiology or conceivably could be an important means to control testosterone synthesis in those clinical cases where this is of benefit. Lastly, it is important to note the magnitude of difference in quantity of Leydig cells and receptors on those Leydig cells of mice and rats. Previously, other investigators have noted the difference in number of Leydig cells between different species (reviewed in 25, 26) and an age-related decrease in the number of Leydig cells and the testosterone synthesis by those Leydig cells (25, 27). Here, we have also observed a decrease in intensity of PGF,,-R staining with aging. In consideration of all these data, the mouse may be a more appropriate animal model system to study in future experiments due to the larger number of Leydig cells, the larger amounts of immunohistochemically detectable PGF*,-R and the higher levels of testosterone synthesis in mouse testis. In conclusion, we have initiated experiments on male
Localization
of PGFr, Receptor in the Mouse Testis
251
reproductive tract tissue with the aim of determining which tissues can respond directly to PGF2,. Our present approach involves the immunohistochemical localization of PGF2, receptors. In the testis, these receptors appear to be confined to Leydig cells. We hypothesize that this may be important in the normal physiology of male sex steroids and that it may potentially be useful in certain clinical situations.
Acknowledgements This work was supported by NIH Grant HD25961-01 to DJO. We also thank N. Hart and C. Mraz for their expert secretarial assistance, and Drs P. Shanley and G. Miller for their critical review of this manuscript.
References 1. Chinoy N J, Sharma J D, Seethalakshmi L, Sanjeevan A G. Effects of prostaglandins on histology of male reproductive organs and fertility in rats. Int J of Fertil 1980; 25: 267-214 2. Eskin B A, Azarbal S, Sepic R, Slate W G. In vitro responses of the spermatozoa-cervical mucus system treated with prostaglandin Fra. Obstet Gynecol 1973; 41: 436-139 3. Cohen M S. Colin M J, Golimbu M. Hot&kiss R S. The effects of prostaglandins on sperm motility. Fertil Stern 1977; 28: 78-85 4. Free M J. Jaffe R A. Effects of prostaglandins on blood flow and pressure in the conscious rat. Prostaglandins 1972: 1: 483-498 5. Einer-Jensen N, Soofi G. Decreased blood flow through rat testis after intratesticular injection of PGFza Prostaglandins 1974 7: 377-382 6. Cavanaugh A H, Farnsworth WE. Receptor sites on human prostate tissue for prostaglandin FZ alpha. Life Sci 1977; 21: 83-92 7. Haour F. Kouznetzova B, Dray F, Saez J M. hCGinduced prostaglandin EZ and Fza release in adult rat testis: role in Leydig cell desensitization to hCG. Life Sci 1979: 24: 2151- 2158 8. Haynes N B. Hafs H D, Waters R J. Manns J G, Riley A. Stimulatory effect of prostaglandin F,, on the plasma concentration of testosterone in bulls. J Endocrinol 1975: 66: 329-338, 9. Kimball F A, Kirton K T, Forbes A D, et al. Serum FSH, LH and testosterone in the male rhesus monkey following prostaglandin injection. Prostaglandins 1979: 18: 117-122 10. Bartke A, Mosto N, Caldwell B X, Behnnan H R. Effects of a cholesterol esterase inhibitor and of prostaglandin Fza on testis cholesterol and on plasma testosterone in mice. Prostaglandins 1973: 3: 97-104 11. Saksena S K, El Safoury S, Bartke A. Prostaglandins EZ and FZa decrease plasma testosterone levels in male rats. Prostaglandins 1973: 4: 235-242 12. Grotjan H E, Heindel J J. Steinberger E. Prostaglandin inhibition of testosterone production induced by luteinizing hormone. dibutyryl cyclic AMP or 3-isobutyll-methyl xanthine in dispersed rat testicular interstitial cells. Steroids 1978; 32: 307-322 13. Fuchs A-R, Chantharaksri U. Prostaglandin Fzu regulation of LH-stimulated testosterone production in rat testis. Biol Reprod 198 1: 25: 492-50 1 14. Yamada K, Ohkura N, Satoh T. Reduction of testosterone levels in rats by prostaglandin Ez, Res Communi Chem Path01 Pharmacol 1985: 47: 153-156 15. Orlicky D J, Miller G J, Evans R M. Identification and purification of a bovine corpora luteal membrane glycoprotein with [3H]prostaglandin Fra binding properties. Prostaglandins Leukotr Essent Fatty Acids 1990: 41: 5161
252
Prostaglandins
Leukotrienes
and Essential Fatty Acids
16. Orlicky D J, Fisher L, Dunscomb N, Miller G J. Immunohistochemical localization of PGF,, receptor in the rat ovary. Prostaglandins Leukotr Essent Fatty Acids 1992; 4633: 223-229 17. Stemberger L A. Hardy P H, Cuculis J J, Meyer H G. The unlabeled antibody enzyme method of immunohistochemistry. Preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-antihorseradish peroxidase) and its use in identification of spirochetes. J Histochem Cytochem 1970; 18: 315-333 18. Orlicky D J, Lieberman R, Williams C, Gerschenson L E. Requirement for prostaglandin Fz,, in 178estradiol stimulation of DNA synthesis in rabbit endometrial cultures. J Cell Physiol 1987; 130: 292-300 19. Albert A. The mammalian testis. In: Young W C, ed. Sex and internal secretions. WiHiams and Wilkins, Baltimore, MD, 1961: 305-365 20. Chowdhury M, Tcholakian R, Steinberger E. An unexpected effect of oestradiol-17pon luteinizing hormone and testosterone. J Endocrinol 1974; 60: 375-376
21. Bartke A, Williams K I H. Dalterio S. Effects of estrogens on testicular testosterone production in vitro. Biol Reprod 1977; 17: 645649 22. Abney T 0. A cytoplasmic estradiol receptor in rat testicular tissue Endocrinol 1976; 99: 555- 563 23. Lin T, Chen G C, Murono E P, Osterman J, Nankin H R. Estradiol receptors of two distinct populations of Leydig cells. Steroids 1982; 40: 53-63 24. Terakawa N, Aono T, Matsumoto K. Presence of two types of estrogen binding sites in mouse testis. J Steroid Biochem 1985; 22: 147-150 25. deKretser D M, Kerr J B. The cytology of the testis. In: Knobil E. Neil1 J D, eds. The physiology of reproduction. Raven Press. New York, 1988: 837-932 26. Setchell B P, Brooks D E. Anatomy, vasculature, innervation, and fluids of the male reproductive tract. In: Knobil E, Neil1 J D, eds. The physiology of reproduction. Raven Press, New York, 1988: 753-836 27. Miller A E, Reigle G D. Aging effects of hypothalamic catecolamine and testosterone secretion in the male rat. Fed Proc 1975: 34: 303 (Abstract #481)
