THE ANATOMICAL RECORD 226:320-327 (1990)

Endorphin Suppresses FSH-Stimulated Proliferation of Isolated Neonatal Sertoli Cells by a Pertussis Tox in- Sensit ive Mechanism JOANNE M. ORTH AND ROSEMARIE BOEHM Department of Anatomy, Temple University School of Medicine, Philadelphia, Pennsylvania 19140

ABSTRACT During perinatal development, when the size of the Sertoli cell population is determined, Leydig cells produce p-endorphin, a peptide which may interact with Sertoli cells to modify their FSH-responsiveness, as suggested by our previous work. The goal of the present study was first, to test directly the possibility that p-endorphin modifies the proliferative response of neonatal Sertoli cells to FSH, and second, to gain information on a mechanismb) involved in any observed effect. We treated isolated 6-day-old Sertoli cells with FSH or vehicle in vitro and measured their incorporation of exogenous, radiolabeled thymidine with quantitative autoradiography. After 2 days in culture with FSH, we detected a 10-fold increase in the rate of Sertoli cell proliferation. The level of cell division in these FSH-treated cultures was identical to that in other cultures exposed to cAMP under similar conditions. In addition, inclusion of P-endorphin 3 h r prior to FSH or cAMP decreased the effect of the hormone by 50% but left the cAMP response unchanged. Thus, p-endorphin acts on isolated, neonatal Sertoli cells at a point prior to intracellular production of cAMP to suppress their response to FSH. When other cultures were treated with pertussis toxin, a blocker of intracellular GTPbinding proteins such as Gi, before sequential addition of endorphin and FSH, the effect of 6-endorphin on FSH-responsiveness was abolished. Moreover, when other cultures were exposed to pertussis toxin in the absence of endorphin, followed by FSH, their response to the hormone was unchanged. Thus, P-endorphin apparently modifies the proliferative response of neonatal Sertoli cells to FSH via a mechanism involving one or more G proteins. These observations, along with our previous data showing enhanced Sertoli cell division in vivo in the presence of a n opiate blocker, point to the existence of endorphin-mediated communication between Leydig and Sertoli cells during perinatal development and provide new evidence suggesting that paracrine mechanisms modify Sertoli cell function during perinatal development, when the size of this population is established. The size of the Sertoli cell population in rats is of critical importance in maintaining normal output of sperm in adults, as evidenced by our findings in Sertoli cell-depleted rats (Orth et al., 1988). Following neonatal treatment with a n anti-mitotic drug, these rats possess lowered numbers of Sertoli cells at maturity. Moreover, although germ cells in neonates are nonproliferative and hence unaffected by the anti-mitotic drug, testes of these rats a s adults display a drop in numbers of spermatids that parallels the decrease in number of Sertoli cells. Hence, a n important quantitative relationship exists between Sertoli and germ cells in the adult testis. Data from our laboratory (Orth, 1982) and that of others (Steinberger and Steinberger, 1971) indicate that Sertoli cells of rats proliferate only during prenatal and early postnatal life, with cessation of division occurring between 10 and 21 days of age. Thus, the perinatal period of testicular development when these cells divide is critical in establishing a Sertoli cell population of a size adequate to insure produc0 1990 WILEY-LISS. INC

tion of normal numbers of sperm at adulthood. As a result, factors that modify Sertoli cell proliferation during perinatal life are likely to be of great importance in insuring fertility of the adult. In previous studies both in vivo and in vitro, we have shown that FSH stimulates Sertoli cells of perinatal rats to divide via a CAMP-mediated process (Orth, 1984) and that the level of Sertoli cell proliferation can be modified during development by altering pituitarytesticular feedback mechanisms. For example, when neonates are hemi-castrated the rate of Sertoli cell division in the remaining testis is elevated in response to a rise in circulating FSH (Orth et al., 1984). Substan-

Received March 8, 1989; accepted May 25, 1989. Data included in this report were presented at the joint meeting of the American Society for Cell Biology and the American Society for Biochemistry and Molecular Biology, San Francisco, CA, January 1989.

ENDORPHIN, FSH, AND SERTOLI CELL PROLIFERATION

tial evidence from other laboratories also indicates that other, intratesticular mechanisms exist, at least in adults, through which the function of testicular cells, including Sertoli cells, can be modified (Sharpe, 1984; Bardin e t al., 1988). One possible route of paracrine regulation of Sertoli cells involves Leydig cells, shown conclusively to be a n intratesticular source of POMCderived peptides such as 0-endorphin throughout fetal and postnatal life (Pintar et al., 1984; Shaha et al., 1984; Bardin e t al., 1987). Several lines of evidence suggest that the Sertoli cell may be a target of endorphin. These include our previous demonstration that interfering with the action of endogenous P-endorphin in testes of fetal or newborn rats, either in vivo or in organ culture, results in suppression of FSHstimulated proliferation of Sertoli cells (Orth, 1986). This observation, along with those pointing to the Leydig cell as a source of endorphin, suggests that endorphin-mediated interaction may occur between Leydig and Sertoli cells during development and that such interaction may modify the responsiveness of Sertoli cells to FSH. Thus, communication between the interstitial and seminiferous compartments of the maturing testis could play a n important role in determining the size of the Sertoli cell population, and therefore the eventual output of sperm, in adult rats. The aim of the present study was to probe further the relationship between FSH-stimulated division of Sertoli cells and p-endorphin. Sertoli cells were isolated from newborns and their rate of proliferation in vitro was studied in the presence or absence of FSH, with or without exogenously added endorphin. Since cells were isolated from neonates, prior to development of SertoliSertoli junctional complexes, it was first important to characterize the makeup of the cultures prior to their use in these studies in order to verify that Sertoli cells were indeed the major component. Therefore, incorporation of 3H-thymidine into cell nuclei and quantitative autoradiography were used as a measure of Sertoli cell proliferation under different conditions. In addition, to test the possible involvement of GTP-binding proteins in any observed effect of endorphin on FSHstimulated proliferation, pertussis toxin was included in some experiments. The resulting data, coupled with our previous findings from studies in vivo, strongly suggest t h a t p-endorphin of Leydig cell origin acts directly on neonatal Sertoli cells to suppress their proliferative response to FSH and that this effect involves interaction of endorphin with GTP-binding proteins in these cells. Thus, p-endorphin-mediated communication between Leydig and Sertoli cells apparently provides a paracrine route through which Leydig cells may modify the hormonal response of Sertoli cells during perinatal development. MATERIALS AND METHODS Sertoli Cell Cultures

For each set of cultures, 20 male pups 6 days old (day of birth = day 1) were killed by decapitation; their testes were removed under aseptic conditions and decapsulated in sterile, ice-cold Dulbecco's Minimum Essential Medium (MEM; Gibco). To isolate Sertoli cells, the testicular tissue was minced and treated further according to the method of Rong-Xi et al. (19871, with

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minor modifications. In brief, the minced tissue was incubated with gentle shaking for 20 min in 30 ml of MEM containing a mixture of 0.1% hyaluronidase (Sigma), 0.1% collagenase (Gibco), and DNAase (Sigma; 0.01 mg/30 ml) in MEM at 37"C, followed by two washes in fresh MEM. After the enzyme treatment and each wash, fragments of seminiferous cords were allowed to settle on ice a t unit gravity for 15 min. Following the last wash, the resulting fragments were incubated at 37°C for 30 min each in 20 ml collagenase (0.1% in MEM) + DNAase twice more, followed by a final rinse in fresh MEM. The resulting fragments were then dispersed into single cells by a brief treatment (approximately 2 min) in Ca-Mg-free Hank's buffer containing 0.05% trypsin (Gibco) and 6 x 10-4 M EDTA. After sedimenting at approximately 200g and washing with BSA-trypsin inhibitor (Sigma; 0.65% and 0.05%, respectively), the resulting cells were resuspended in a small volume of plating medium (see below). For each set of cultures, cell viability was evaluated by exposing a n aliquot of the single cell suspension to trypan blue and determining the yield of dyenegative cells in a hemocytometer. Cells were cultured a t 37°C in a 5% COz atmosphere in four-chamber tissue culture slides (Lab-Tek) previously coated with Matrigel (Collaborative Research); the latter was diluted 1 : l with MEM, applied as a thin coat to the slides and allowed to gel for 2-3 h r a t 32°C prior to use. Two million cells were plated in each chamber (1 x 104/mm2)in serum-free Eagle's D-val MEM (Gibco) supplemented with Na Pyruvate (1mM) and non-essential amino acids (0.1 mM), with fungizone (2.5 pg/ml) and penicillin (100 U/ml)-streptomycin (100 pg/ml) also included. The next morning, approximately 18 h r after plating, all chambers were rinsed in warm medium and fresh medium was added. To determine the proportion of Sertoli cells to other testicular cells (e.g., peritubular and Leydig cells) in these preparations, several approaches were applied to representative chambers. First, some cultures were evaluated morphologically by processing them for electron microscopy, a s described below. Second, the ability of cells in other chambers to respond to cAMP with a change in shape was determined by adding db cAMP (0.5 pM) or vehicle for 3-18 hr, followed by fixation and inspection of the cells with phase contrast microscopy. Finally, several chambers were evaluated with cytochemistry as previously described (Orth and Weisz, 1980) for the presence of cells displaying activity of either 3P-hydroxysteroid dehydrogenase, a Leydig cell marker, or alkaline phosphatase, a n indicator of peritubular cells (Chapin et al., 1987). Studies of Sertoli Cell Proliferation In Vitro

To evaluate the effect of P-endorphin on Sertoli cell division following exposure of cells to FSH or CAMP, the following treatment grdups were established: first, some cultures received FSH (oFSH-17; 1 pg/ml) from either day 2 of the experiment onward or for only the final 24 h r of culture, with and without addition of endorphin (1 pg/ml) 3 h r before FSH. Other cultures received db cAMP (0.5 pM), again with and without prior addition of endorphin, either from day 2 onward or for only the last day of culture. To probe the mechanism involved in any observed

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effect of p-endorphin, additional chambers first received pertussis toxin (1 pg/ml), a blocker of GTPbinding proteins, including Gi (Murayama and Ui, 1983);either p-endorphin or vehicle was added to these chambers 3 h r later, followed by FSH, CAMP,or vehicle after another 3 h r period. Additional cultures received P-endorphin or pertussis toxin alone or in combination, but no FSH or CAMP.Controls received only vehicle a t appropriate times. All cultures were maintained for a total of 4 days, with the various agents present from the time of addition onward. For chambers that received hormone andlor peptide from days 2 through 4, a n agentb) was freshly added on day 3 when medium was changed. In all chambers, 3H-thymidine (New England Nuclear; 6.7 Ci/mmol; 1 pCi/ml final concentration) was included for the final 24 h r in vitro. At the end of the 4th day, cultures were fixed a s described below and subjected to autoradiography as detailed previously (Orth, 1982). In brief, slides were coated after fixation with undiluted Kodak NTB-3 emulsion and allowed to expose in darkness for 7 days. At the end of this period, the cultures were photographically developed, stained with methylene blue, and viewed with conventional light microscopy for quantitation. Proliferative nuclei were heavily labeled by 3H-thymidine and, after this length of photographic exposure, were overlain by silver grains and easily distinguished from those surrounding nuclei that had not incorporated label. Quantitation and Statistical Analysis

These experiments were carried out on three separate occasions, with a different cell isolation providing the cultures each time. For each trial, cell proliferation was measured in all treatment groups a s follows: cultures were examined a t 100 x magnification; 2,0003,000 cell nuclei were chosen in each chamber in a non-random manner to insure against viewer bias and were scored a s labeled or unlabeled by 3H-thymidine. The data for each chamber were expressed as the percentage of cells counted that had incorporated label into their nuclei. Thus, three or four chambers were studied and a total of 6,000 or more cells were quantified for each treatment. The final data were expressed as the mean percentage of cells labeled ( & SEM) for the various groups. A one-way analysis of variance was then used to determine whether differences existed among the groups, and these differences were subsequently located with a Newman-Keuls test. Electron Microscopy

Plastic chambers were removed from the culture slides and cells were processed in situ for electron microscopy a s follows. After fixing for 30 min a t 4°C in 2.5% glutaraldehyde-0.1M Na cacodylate, pH 7.4, containing 5% sucrose, the cultures were post-fixed in 1% osmium tetroxide reduced with 1.5%K ferrocyanide for 30 min and then mordanted in 1% tannic acid in O.1M Na cacodylate for 30 min at 4°C. Following subsequent dehydration through 100% ethanol, the cultures were infiltrated with Epon-Araldite. Beem capsules containing unpolymerized plastic were then inverted over the cells and the slides were placed overnight at 60°C. The next day, capsules containing polymerized plastic with embedded cells were removed from the slides by brief

Fig. 1. An electron micrograph of cells isolated from 6-day-old pups, fixed the morning after plating, and sectioned parallel to the surface of the chamber. The great majority of cells in these cultures possessed irregular, indented nuclei with single nucleoli, elongated mitochondria, and occasional lipid droplets, characteristics consistent with those of Sertoli cells. x 6,630.

immersion of the entire slide in liquid N P . Thin sections were cut parallel to the surface of the cultures with a Reichert Ultracut E microtome, post-stained in uranyl acetate and Reynold's lead citrate, and viewed and photographed with a Philips 300 electron microscope. RESULTS Characterization of Cultures

Isolated cells adhered to and spread upon the underlying Matrigel substrate within 30-60 min after plating to form confluent cultures. Figure 1is a representative ultrastructural view of a culture that was rinsed and fixed the morning after plating; the plane of section was parallel to the surface of the chamber. The great majority of the cells in these cultures had a morphology consistent with that of Sertoli cells, with irregular, often indented nuclei containing some peripheral heterochromatin. Nucleoli, where present in the section, were single and displayed a tripartite morphology characteristic of Sertoli cells. In addition, mitochondria were typically elongated and cytoplasmic liquid droplets were occasionally seen. Peritubular cells, easily identifiable by their rough endoplasmic reticulum con-

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Fig. 2. Cultures incubated on the morning after isolation with either dibutyryl cAMP (b)or vehicle (a) and fixed 3 hr later. Nearly all cells responded to the presence of the cyclic nucleotide with a rapid and dramatic change in shape which was maintained as long as cAMP was present. x 400.

taining copious flocculent material, were encountered on rare occasion. When similar cultures were exposed to dibutyrl cAMP and viewed with phase contrast microscopy, the vast majority of the cells became rounded centrally and displayed elongated peripheral processes, a s shown in Figure 2. This change in cell shape was apparent in most cells by 1 h r and was maintained so long as cAMP was present in the cultures. In addition, less than 5%of the cells in representative cultures displayed reaction product following cytochemical incubation for alkaline phosphatase, a n enzyme characteristic of myoid cells. Finally, to probe the possibility of contamination by Leydig cells, the activity of the steroidogenic enzyme 3p-hydroxysteroid dehydrogenase was also visualized with LM cytochemistry. Virtually no cells reactive for this enzyme were encountered in any of the chambers examined. FSH, p-Endorphin, and Sertoli Cell Proliferation In Vitro

Figure 3 provides typical autoradiographs of cultures that were either untreated or exposed to FSH from day 2 of culture onward, with and without inclusion of pendorphin. Labeled thymidine was present during the final 24 hours of culture for all treatments. While few labeled nuclei were found in unstimulated controls (Fig. 3a), FSH caused a dramatic and obvious increase in the proportion of Sertoli cells t h a t incorporated 3H-thymidine into their nuclei (Fig. 3b). However, when p-endorphin was added 3 h r prior to the hormone, the proliferative response of the cells to FSH was apparently suppressed (Fig. 3c). This qualitative obser-

vation was confirmed when dividing Sertoli cells were quantified in autoradiographs of similarly treated cultures; the final data from all groups studied, expressed as the mean of three trials for each treatment, are given in Figure 4. The level of proliferation in unstimulated control cultures was low, below 2% in all dishes, and unaffected by pertussis toxin alone or pertussis toxin plus endorphin. However, addition of either FSH or cAMP on day 2 resulted in a n approximately 10-fold increase (P

Endorphin suppresses FSH-stimulated proliferation of isolated neonatal Sertoli cells by a pertussis toxin-sensitive mechanism.

During perinatal development, when the size of the Sertoli cell population is determined, Leydig cells produce beta-endorphin, a peptide which may int...
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