Hormonal regulation of gonadotropin receptors and steroidogenesis in cultured fetal rat tests.

Gonadotropic activation of the adult rat testis in vitro and in vivo is followed by down-regulation of luteinizing hormone receptors and decreased androgen responses to subsequent hormonal stimulation. In contrast, treatment of cultured fetal testes with gonadotropins and dibutyryl adenosine 3',5'-monophosphate enhanced steroidogenic responsiveness and did not cause the luteinizing hormone-receptor loss and desensitization that is characteristic of the adult gonad. The analysis of gonadotropin receptors and action in cultured fetal testis cells facilitates developmental studies of gonadal function, and has revealed significant differences in the responses of fetal and adult Leydig cells to gonadotropic regulation.

A novel alphavirus vaccine encoding prostate-specific membrane antigen elicits potent cellular and humoral immune responses.

PURPOSE: Prostate-specific membrane antigen (PSMA) is an attractive target for active immunotherapy. Alphavirus vaccines have shown promise in eliciting immunity to tumor antigens. This study investigated the immunogenicity of alphavirus vaccine replicon particles (VRP) that encode PSMA (PSMA-VRP). EXPERIMENTAL DESIGN: Cells were infected with PSMA-VRP and evaluated for PSMA expression and folate hydrolase activity. Mice were immunized s.c. with PSMA-VRP or purified PSMA protein. Sera, splenocytes, and purified T cells were evaluated for the magnitude, durability, and epitope specificity of the anti-PSMA response. Antibodies were measured by flow cytometry, and cellular responses were measured by IFN-gamma enzyme-linked immunospot and chromium release assays. Cellular responses in BALB/c and C57BL/6 mice were mapped using overlapping 15-mer PSMA peptides. A Good Laboratory Practice-compliant toxicology study was conducted in rabbits. RESULTS: PSMA-VRP directed high-level expression of active PSMA. Robust T-cell and B-cell responses were elicited by a single injection of 2 x 10(5) infectious units, and responses were boosted following repeat immunizations. Anti-PSMA responses were detected following three immunizations with 10(2) infectious units and increased with increasing dose. PSMA-VRP was more immunogenic than adjuvanted PSMA protein. Responses to PSMA-VRP were characterized by Th-1 cytokines, potent CTL activity, and IgG2a/IgG2b antibodies. T-cell responses in BALB/c and C57BL/6 mice were directed toward different PSMA peptides. Immunogenic doses of PSMA-VRP were well tolerated in mice and rabbits. CONCLUSIONS: PSMA-VRP elicited potent cellular and humoral immunity in mice, and specific anti-PSMA responses were boosted on repeat dosing. PSMA-VRP represents a promising approach for immunotherapy of prostate cancer.

A Perspective on the Evolving Story of PSMA Biology, PSMA-Based Imaging, and Endoradiotherapeutic Strategies.

In this review, we cover the evolution of knowledge on the biology of prostate-specific membrane antigen (PSMA) and its translation to therapy. The usual key to discovery is a realistic model for experimentation and for testing a hypothesis. A realistic model is especially needed in the case of the human prostate, which differs significantly from the prostate of species often used as research models. We will emphasize the genetic characterization of PSMA, the nature of the PSMA protein, and its role as a carboxypeptidase, with differing important substrates and products in different tissues. We give special prominence to the importance of PSMA as a target for imaging and therapy in prostate cancer and its underdeveloped role for imaging and targeting the neovasculature of tumors other than prostate cancer. Lastly, we bring attention to its importance in other nonprostatic tissues.

2-5A ligands--a new concept for the treatment of prostate cancer.

Several potent prostate specific membrane antigen (PSMA) inhibitors have been described recently. We generated a PSMA-specific 2-5A ligand called RBI 1033 by linking 2-5A to the N-acetylaspartylglutamate (NAAG)-based inhibitor ZJ-24. We measured the inhibitory activity of RBI 1033 to the folate hydrolase activity of PSMA. Amazingly, we found that compared to ZJ-24 (IC50 = 53.9 nM), RBI 1033 was more than 10 times more potent (IC50 = 4.78 nM) as a folate hydrolase inhibitor, while SMCC 2-5A lacking the ZJ-24 part, did not show much activity (IC50 = 1974 nM). Also, RBI 1033's affinity to PSMA was found to be 10 times higher than ZJ-24 itself.

Novel role of prostate-specific membrane antigen in suppressing prostate cancer invasiveness.

Prostate-specific membrane antigen (PSMA), a type II transmembrane glycoprotein, is overexpressed in prostate cancer. PSMA is a unique cell surface marker, negatively regulated by androgen and extensively used for imaging of hormone refractory carcinomas and metastatic foci. PSMA is a carboxypeptidase with two important enzymatic functions, namely, folate hydrolase and NAALADase. PSMA also exhibits an endocytic function, in which it spontaneously recycles through endocytic vesicles. PSMA is overexpressed at various stages of prostate cancer, including androgen-sensitive and -independent disease, increased in expression with early relapse after therapy. We have used in vitro invasion assays to explore the possible role of PSMA in the metastasis of prostate cancer cells. Androgen-dependent prostate cancer lines, which express PSMA endogenously (e.g., LNCaP, MDA PCa2b, and CWR22Rv1) are less invasive compared with androgen-independent PC3 or DU145 cells, neither of which expresses PSMA. Ectopic expression of PSMA in PC3 cells reduced the invasiveness of these cells, suggesting that this reduction in the invasion capability of PSMA-expressing cells is due to PSMA expression and not to intrinsic properties of different prostate cancer cell lines. Furthermore, knockdown of PSMA expression increased invasiveness of LNCaP cells by 5-fold. Finally, expression of PSMA mutants lacking carboxypeptidase activity reduced the impact of PSMA expression on invasiveness. Thus, it seems that the enzymatic activity is associated with the effect of PSMA on invasiveness.

Should Low Molecular Weight PSMA Targeted Ligands Get Bigger and Use Albumin Ligands for PSMA Targeting?

Prostate Specific Membrane Antigen (PSMA) is strongly expressed in prostate cancer. Recently a number of low-molecular-weight inhibitors have demonstrated excellent PSMA targeting activity for both imaging as well as Lutecium-177 radiotherapy in human trials. The paper by Choy et al raises the question of whether we can further increase the effectiveness of PSMA targeted therapy by adding an albumin-binding entity to low-molecular-weight agents.

Ontogeny of pituitary-gonadal interactions: current advances and controversies.

The different compartments of the fetal hypothalamic-pituitary-gonadal axis, the hypothalamus, anterior pituitary, and gonads, probably start their embryonic development independently, and become fully interactive as the last link o f their maturation. The developing hypothalamic-pituitary-gonadal axis offers a good model for studies on the mechanisms of regulation of fetal hormonal systems. It is evident that fetal hormonal functions are not the same as those of the adult on a smaller scale, but that there are fundamental differences between the fetus and adult in basic features of the mechanisms o f reproductive hormone action.

The rat 78,000 dalton glucose-regulated protein (GRP78) as a precursor for the rat steroidogenesis-activator polypeptide (SAP): the SAP coding sequence is homologous with the terminal end of GRP78.

Based on the striking sequence identity between the amino acid sequence of rat steroidogenesis-activator polypeptide (SAP) and the carboxyl terminus of the 78,000 dalton glucose-regulated protein (GRP78), the precursor-product relationship between GRP78 and SAP was investigated in Leydig cells. Immunoblot analysis with peptide antibodies specific for GRP78 and SAP showed that the putative SAP precursor is also immunoreactive with the anti-GRP78 antibody. Genomic blot hybridizations further revealed that GRP78 is neither rearranged nor amplified in the H-540 Leydig cell tumor, the original source for SAP. Further, there appears to be a single copy of the SAP coding sequence within the rat genome. This sequence resides within the last exon of GRP78. Our observations support the hypothesis that, in steroidogenic cells, SAP is likely to be derived from posttranslational processing of a very minor fraction of GRP78.

A novel role for testicular descent; temperature-dependent induction of pertussis toxin-sensitive Gi protein function in postnatal rat Leydig cells.

The role of temperature and testicular descent in postnatal appearance of inhibitory guanine nucleotide-binding regulatory protein (G(i)) function was studied in the rat testis. Dispersed testicular cells of 5-day-old rats were incubated for 24 h at 32 or 37 C, then for another 24 h at the same temperatures in the presence and absence of pertussis toxin (PT; 100 micrograms/liter), and finally for a third 24-h period with cholera toxin (CT; 500 ng/liter) with or without PT. At both temperatures, PT treatment significantly (P < 0.05) increased the CT-stimulated cAMP output, but had no effect on basal cAMP production. When testosterone (T) production, as an indicator of Leydig cell function, was measured in the same incubation, CT-stimulated T production was greater at 32 C, but PT had no effect at either temperature. A similar finding was made when hCG (10 micrograms/liter), instead of CT, was used as the stimulus of T production. Hence, a functional G(i) protein is present in seminiferous tubules of 5-day-old testes cultured for 3 days at 32 and 37 C, but not in Leydig cells. We then examined the effects of longer exposure of 5-day-old testes to the two temperatures. After culture for 7 days with 0.1 microgram/liter ovine LH, the presence of PT at 32 C significantly (P < 0.01) enhanced CT-stimulated T production during the last 24 h of culture, but the PT effect was not observed when the culture was carried out at 37 C. Hence, G(i)-mediated modulation of Leydig cell function appears to require several days of induction at the lower temperature of 32 C. As the postnatal descent also changes the ambient testicular temperature, we next studied whether this event alters the G(i) protein function of Leydig cells. Five-day-old rats were rendered bilaterally cryptorchid or sham operated, and studied after 12 days. Testis weights did not differ between the abdominal and scrotal testes. In contrast, the basal and hCG-stimulated rates of T production were significantly (P < 0.01-0.05) higher in the scrotal testes. When dispersed cells of the scrotal and abdominal testes were incubated for 24 h at 37 C in the presence of CT with or without PT, enhancement of T production by PT was only observed in cells of the scrotal testes.(ABSTRACT TRUNCATED AT 400 WORDS)

The effect of gonadotrophins on the fetal and neonatal rat testis.

Testosterone formation was measured in fetal and neonatal (2 h to 20 days) rat testes incubated with and without 100 IU human chorionic gonadotrophin (hCG). At all ages studied, testosterone production was significantly elevated in the presence of hCG. Neonatal testes showed an absolute drop in testosterone production with increasing age, but the percentage increase above baseline produced by hCG stimulation leads to the speculation that this decrease is due to a relative reduction in the number of Leydig cells with increasing age rather than to a lowered sensitivity to hCG stimulation.

Ontogeny of gonadotropin receptors in the fetal and neonatal rat testis.

To evaluate the extent to which LH receptors may participate in the regulation of testosterone production by the fetal and neonatal rat testis, the gonadal content of LH receptors was determined and correlated with the gonadal content of testosterone and other steroids at daily intervals throughout the period of sexual differentiation and early neonatal life. Also, to determine if FSH has a potential role during fetal life, FSH receptors were measured from 14.5 until 21.5 days of gestation, just before birth. LH receptors were first detected in the fetal rat testis at 15.5 days of gestation, at a concentration of 0.072 +/- 0.016 fmol/gonad. This is the time at which previous studies have shown a biological response to LH stimulation with increases in cAMP and testosterone production. LH receptor content remained constant from 15.5-17.5 days of gestation and then increased sharply at 18.5 days of gestation to 0.706 +/- 0.046 fmol/gonad, coincident with a surge in the testosterone content of the fetal testis. LH receptor content continued to rise until birth, reaching a maximum level of 2.00 +/- 0.24 fmol/gonad at 21.5 days of gestation, and did not significantly differ from this value through 5 days after birth, whereas intratesticular testosterone content decreased after birth. FSH receptors could be measured from 17.5 days of gestation, but remained very low through 19.5 days of fetal life. At 20.5 days, FSH receptor levels began to rise sharply and continued to increase through 21.5 days of gestation, just before birth. These data show that LH receptors are measurable at the time when the fetal testis becomes responsive to LH stimulation. It is possible that the increase in LH receptors may act as a mechanism for amplification of the stimulatory effect of low circulating LH levels on the fetal testis being concomitant with the intratesticular testosterone increase at the same stage. The decrease in testosterone production after birth cannot be attributed to a decrease in LH receptors in the neonatal gonad. Since 5 alpha-reduced androgens increase after birth, other possibilities, such as alternative steroid pathways, are a more likely explanation for the decrease in testosterone content. The presence of FSH receptors in the fetal testis imply that this hormone may have a function in the developing gonad, particularly in the last 2 days of fetal life.

A new method for measurement of the conversion ratio of thyroxine to triiodothyronine in euthyroid man.

A new method for the measurement of the conversion ratio (CR) of T4 to T3 in euthyroid man is described. In contrast to previously described studies, this investigation relied on sampling of urine rather than plasma after isotopic labeling of the study subject. The CR value determined in six euthyroid men was 0.482 +/- 0.014. Thus, approximately half of the daily T4 production is converted to T3 as determined by this method. A major advantage of this technique is its reproducibility, as demonstrated by the low coefficient of variation of 6.8% in the study group, which is not significantly different from the 6.7% coefficient of variation for replicate determinations on the same sample. Thus, this method may be useful tool in comparing the CR of T4 to T3 in man under varying conditions even if only small differences in conversion efficiencies exist between groups. One apparent discrepancy observed in the present study is that the calculated T3 produced from the conversion of T4 exceeded the simultaneously calculated daily T3 production rate measured in blood. The cause of this discrepancy is presently unknown, but may represent T3 produced from T4 by renal or extrarenal sources which is excreted without contributing to the T3 blood production rate. However, the reproducibility of the method and the smaller amounts of labeled isotope required show promise that this technique may be useful in assessing T4 to T3 conversion in a variety of altered metabolic states.

The significance of partial suppressibility of serum thyroxine by triidothyronine administration in euthyroid man.

Recent evidence indicates that triiodothyronine (T3) administration may not completely inhibit normal thyroid secretion. To further corroborate this observation, measurement of serum T4-RIA concentrations was performed on 15 normal controls (10 men, 5 women; ages 20-42) who were placed on 100 mug of T3 daily for a 5-week period. Decrements of 53%, 36%, and 28% from the baseline T4-RIA were noted at weeks 1, 2, and 3 respectively. At 3 weeks a nadir T4-RIA of 2.5 mug/100 ml was reached which did not significantly differ from the 4th (2.9 mug/100 ml) and 5th weeks (2.6 mug/100 ml). Further, seven euthyroid patients who had received replacement thyroid hormone for 1-16 were switched to T3 (75-100 mug/day) for 28 days. At the end of this period, their mean T4-RIA was 2.6 mug/100 ml. Similar T3 treatment studies were performed on 20 primary hypothyroid patients. After 4 weeks of T3 all 20 patients displayed a T4-RIA below the limits of assay detectability (less than 0.625 mug/100 ml) while all euthyroid subjects had values greater than 1.2 mug/100 ml. Suppression of T4-RIA with T3 was also noted in 4 patients with pituitary and 2 patients with hypothalamic hypothyroidism. Three days after cessation of T3 treatment in normal subjects, no significant rise in mean T4-RIA was seen (2.3 mug/100 ml). Subsequently, T4-RIA rose to 4.5 mug/100 ml on day 7 and 6.7 mug/100 ml on day 10 (74% of the presuppression value) in normals. A similar rise to 7.9 mug/100 ml 10 days after withdrawal from T3 was noted in the euthyroid subjects who had received long-term thyroid hormone replacement. In contrast, all primary hypothyroid patients had either a minimal or nondetectable elevation in T4-RIA while demonstrating a marked rise in TSH 10 days after T3 withdrawal. An absent or impaired rise in T4-RIA after T3 withdrawal was also noted in patients with pituitary and hypothalamic hypothyroidism. These observations indicated: 1) There is continued thyroidal T4 secretion in euthyroid subjects receiving 100 mug of T3 daily. 2) The hypothesis is advanced that an intact hypothalamic-pituitary-tyhroid axis may be required for continued T4 secretion while on T3. 3) The duration of prior suppression with thyroid hormone medication does not appear to influence this phenomenon.

Urinary immunoprecipitation method for estimation of thyroxine to triiodothyronine conversion in altered thyroid states.

A new method is described for the estimation of T4 to T3 conversion in man and is applied to the study of hyperthyroid and hypothyroid clinical states. The method employs simultaneous iv injection of [125I]T4 and [131I]T3 with isolation of the labeled T3 tracers in 4- to 8-day pooled urine samples by a combination of solvent extraction, desalting, and immunoprecipitation procedures. Using [131I]T3 as a recovery standard, the T4 to T3 conversion ratio was found to be 0.470 +/- 0.011 in euthyroid subjects. This confirmed our earlier findings of 0.482 +/- 0.014 using a paper chromatographic method and nonsimultaneous isotope administration. The conversion ratio was increased in hypothyroidism to 0.535 +/- 0.011 (P less than 0.02) and decreased in hyperthyroidism to 0.415 +/- 0.009 (P less than 0.01). These changes parallel the fraction of the radioiodine collected in the urine for both T4 and T3; normal values are 77 +/- 4% for T4 and 76 +/- 4% for T3, values in hypothyroidism are 79 +/- 1% for T4 and 79 +/- 3% for T3, and values in hyperthyroidism are 58 +/- 3% for T4 and 58 +/- 5% for T3 (P less than 0.01). These findings indicate that 1) urinary T4 to T3 conversion values are highly reproducible in euthyroid as well as hyperthyroid and hypothyroid states; 2) the reduction in T4 to T3 conversion in hyperthyroidism probably reflects increased T4 disposal by nondeiodinative pathways and possibly the reverse in hypothyroid states; and 3) since urinary T4 to T3 conversion values in euthyroid subjects exceeded all reported conversion values in blood, there may be an alternate pathway of T3 production and disposal which is not reflected in the blood T3 production rate.

Steroidogenic enzyme activities, morphology, and receptor studies of a testicular adrenal rest in a patient with congenital adrenal hyperplasia.

Steroid-secreting tumors of the testis have generally been considered to be of Leydig cell origin. Testicular tumors in patients with congenital adrenal hyperplasia have been thought to be adrenal rests, but no conclusive evidence supporting the hypothesis has been presented. We report a morphological and biochemical analysis of a patient with 21-hydroxylase deficiency who developed bilateral nodular hyperplasia of steroid-secreting tissue within the testis, despite suppression therapy with both exogenous glucocorticoids and testosterone. The tissue was formed of confluent nodules of homogenous cells. Electron microscopy showed the cells to have abundant smooth endoplasmic reticulum, well developed Golgi apparatus, and mitochondria with predominantly tubular cristae, features characteristic of steroid-secreting cells of adrenocortical origin. Crystals of Reinke were not observed. Functional studies in vivo showed a marked response to ACTH infusion, with 17-hydroxyprogesterone rising from 56 to 13,500 ng/mL, cortisol from less than 2 to 19 micrograms/dL, and testosterone from 369 to 629 ng/dL, with an attendant increase in testicular size and pain over 48 h. Receptor studies in vitro revealed no gonadotropin receptors, but abundant angiotensin-II receptors. Enzyme activity analysis in vitro showed undetectable 21-hydroxylase activity and an enzyme profile consistent with adrenocortical cells rather than Leydig cells. Based on these morphological and biochemical findings, we conclude that the nodular steroidogenic tissue that replaced this patient's testes was of adrenal origin. The study documents for the first time the development of adrenocortical tumors from adrenal rest tissue within the testis.

Prolactin localization, binding, and effects on peroxidase release in rat exorbital lacrimal gland.

Prolactin immunoreactivity has been detected in human tears and in lacrimal glands, and it has been suggested that this hormone might be a modulator of lacrimal secretion as well as a component of lacrimal gland fluid. The present study was designed to confirm the immunocytochemical localization of prolactin in the rat lacrimal gland, to determine the source of the prolactin, and to evaluate the acute effects of prolactin on lacrimal secretory function. We have confirmed that prolactin-like immunoreactivity is present in secretory vesicles of acinar cells of male and female Sprague-Dawley rats. Prolactin message was present at detectable levels in RNA extracts of lacrimal glands from males, indicating that at least a component of the prolactin-like immunoreactivity was the product of synthesis within the lacrimal glands. Crude membrane fractions from acini isolated from males bound 43.1 +/- 3.2 femtomoles prolactin/mg protein (mean +/- standard error of the mean; n = 6), which was significantly (P less than 0.01) more than comparable fractions from females (15.4 +/- 2.4 fmoles/mg protein, n = 6). Preincubating membranes at 65 degrees for 20 min to release endogenous ligands increased prolactin binding to 84.8 +/- 20.8 fmoles/mg protein for males and 63.8 +/- 17.4 fmoles/mg protein for females (P greater than 0.1), suggesting that, on average, similar numbers of receptors are expressed in acinar cells of male and female rats but a larger fraction of the receptors is occupied by endogenous prolactin-like peptides in females. Because prolactin binding triggers prolactin receptor internalization in various cell types, we propose that the prolactin-like immunoreactivity in lacrimal acinar cells of females has been accumulated from the circulation, while the immunoreactivity seen in males results, at least in part, from de novo synthesis. Ovine prolactin at concentrations of 10-20 ng/ml inhibited carbachol-induced peroxidase release by 19.6% +/- 6.9% (n = 8, P less than 0.02) but failed to alter peroxidase release in the absence of carbachol. These observations suggest that prolactin might function as an endocrine, paracrine, or autocrine modulator in the lacrimal gland.

Hypophysectomy-induced regression of female rat lacrimal glands: partial restoration and maintenance by dihydrotestosterone and prolactin.

PURPOSE: Previous studies have implicated androgens and one or more as yet unknown pituitary or pituitary-dependent factors in the regulation of certain lacrimal gland functions. Many observations suggest that prolactin (PRL) might well be one of these factors. This study was designed to determine the effect of hypophysectomy on biochemical markers of exorbital lacrimal gland secretory capacity and to determine the extent to which dihydrotestosterone (DHT) and prolactin reverse these changes. METHODS: Female rats were hypophysectomized and, 5 days later, were treated for 2 days with DHT (0.25 or 1 mg/kg), PRL (1 or 5 mg/kg), combinations of the low or high doses of DHT and PRL, or vehicle only. The animals were killed, and crude membrane fractions were isolated from their lacrimal glands. An untreated group served as control. RESULTS: Lacrimal glands atrophied rapidly after hypophysectomy, losing 40% of their total and membrane-associated protein and 50% of their total DNA within 5 days. Total Na+,K(+)-ATPase and acid phosphatase activities and beta-adrenergic receptor number were decreased by half, whereas alkaline phosphatase activity and muscarinic cholinergic receptor number were reduced by 25% to 30%. DHT treatment increased total DNA above control values; it partially restored the amount of protein in the gland, the Na+,K(+)-ATPase and acid phosphatase activities, and the beta-adrenergic receptor number; and it fully restored the alkaline phosphatase activity. Prolactin treatment partially restored the amount of protein in the gland and the Na+,K(+)-ATPase activity; it fully restored the alkaline phosphatase activity and cholinergic receptor number; but it had no effect on the acid phosphatase activity or the beta-adrenergic receptor number. The high dose of DHT reduced the increase in cholinergic receptor number elicited by PRL. The high dose of PRL reduced the increases of total Na+,K(+)-ATPase and acid phosphatase elicited by DHT. CONCLUSIONS: These findings suggest that DHT and PRL exert general trophic actions on the lacrimal gland and specifically on lacrimal Na+,K(+)-ATPase, acid phosphatase, and neurotransmitter receptors. They also suggest that excessive levels of either hormone may be deleterious to secretory function. Because sex hormone levels are prone to wide fluctuations in women, our results also suggest a plausible hypothesis to account for the greater incidence in women of lacrimal insufficiency.

Androgen influence on lacrimal gland apoptosis, necrosis, and lymphocytic infiltration.

PURPOSE: Previous studies have shown that ovariectomy and hypophysectomy cause regression of the lacrimal gland and have implicated androgens as trophic hormones that support the gland. The purposes of this study were to test the hypothesis that glandular regression after ovariectomy is due to apoptosis, to identify the cell type or types that undergo apoptosis, to survey the time course of the apoptosis, and to determine whether ovariectomy-induced apoptosis could be prevented by dihydrotestosterone (DHT) treatment. METHODS: Groups of sexually mature female New Zealand White rabbits were ovariectomized and killed at various time periods up to 9 days. Additional groups of ovariectomized rabbits were treated with 4 mg/kg DHT per day. At each time period, sham-operated rabbits were used as controls. Lacrimal glands were removed and processed for analysis of apoptosis as assessed by DNA fragmentation and for morphologic examination. DNA fragmentation was determined using the TdT-dUTP terminal nick-end labeling assay and by agarose gel electrophoresis. Labeled nuclei were quantified by automated densitometry. Sections were also stained for RTLA (rabbit thymic lymphocyte antigen), rabbit CD18, and La antigen. Morphology was evaluated by both light and electron microscopy. RESULTS: The time course of apoptosis exhibited two phases, a rapid and transient phase and a second prolonged phase. A transient phase peaked at approximately 4 to 6 hours after ovariectomy. The values for degraded DNA as a percentage of total nuclear area were 4.29%+/-0.79% and 4.26%+/-0.54%, respectively. The values for sham-operated controls examined at the same time periods were 1.77%+/-0.08% and 0.82%+/-0.21%, respectively. The percentage of degraded DNA at 24 hours after ovariectomy was not different from controls examined at the same interval after sham operation. The percentage of degraded DNA 6 days after ovariectomy was significantly increased (8.5%+/-2.4%), compared with sham-operated animals at the same time period (0.68%+/-0.03%). DNA laddering was more pronounced after ovariectomy. Dihydrotestosterone treatment in ovariectomized rabbits suppressed the increase in DNA degradation. Morphologic examination of lacrimal gland sections indicated that ovariectomy caused apoptosis of interstitial cells rather than acinar or ductal epithelial cells. Tissue taken 4 hours and 6 days after ovariectomy showed nuclear chromatin condensation principally in plasma cells. Increased numbers of macrophages were also evident. Significant levels of cell degeneration and cell debris, characteristic of necrosis, were observed in acinar regions 6 days after ovariectomy. Dihydrotestosterone prevented this necrosis. Increased numbers of RTLA+, CD18+, and La+ interstitial cells were also evident 6 days after ovariectomy. In addition, ovariectomy increased La expression in ductal cells. Dihydrotestosterone treatment prevented the increase in numbers of lymphoid cells and La expression. Dihydrotestosterone also promoted the appearance of mitotic figures in acinar cells and increased the sizes of acini by 43% (P < 0.05). CONCLUSIONS: Glandular atrophy observed after ovariectomy is likely to proceed by necrosis of acinar cells rather than apoptosis. This process begins with an apparent time lag after a rapid phase of interstitial cell apoptosis. These processes are accompanied by increased lymphocytic infiltration. These results suggest that a critical level of androgen is necessary to maintain lacrimal gland structure and function and that a decrease in available androgen below this level could trigger lacrimal gland apoptosis and necrosis, and an autoimmune response. Because apoptotic and necrotic cell fragments may be sources of autoantigens that can be processed and presented to initiate an autoimmune reaction, we surmise that cell death triggered by androgen withdrawal may trigger an autoimmune response such as that encountered in Sjögren's syndrome. (ABSTRACT TRUNCATED)