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The distribution of testin in the female reproductive system of rats throughout the estrous cycle was examined immunohistochemically. In ovarian follicles, immunostainable testin was localized at the junctions between adjacent granulosa cells. During follicular development, immunostainable testin surrounding the granulosa cells increased in every follicle but was reduced drastically when the follicle was undergoing atresia. Testin was also found in the junctions between adjacent germinal epithelial cells that covered the surface of the ovary, at the lower or the lower lateral borders of each cell. In the uterus and oviduct, immunostainable testin was detected only in the luminal and glandular epithelium, where it formed a polygonal network encircling the apical border of the epithelial cells. During the estrous cycle, there was no drastic change in the distribution of testin in the epithelial cells of the ovary. In the vaginal mucosa, testin was found to be localized only at the junction of the epithelial cells on the surface layer of the stratified epithelium ; at different stages of the estrous cycle, distinctive staining for testin could be found at proestrus, metestrus, and diestrus, but not at estrus. It is postulated that testin is a cell junction-associated protein in the female reproductive system.

Leydig and Sertoli cells are both targets of androgen action in the testis. Androgen exerts contrasting effects on the two cell types partially inhibiting steroidogenesis in adult Leydig cell and stimulating adult Sertoli cell functions required to support spermatogenesis. The developmental changes in the messenger RNA (mRNA) levels of androgen receptor (AR) also differ between Leydig and Sertoli cells, with Leydig cell AR mRNA being highest on day 35 postpartum, whereas Sertoli cell AR mRNA levels are highest on day 90. The purpose of the present study was to determine if the concentrations of AR in Leydig and Sertoli cells are differentially regulated during development using quantitative immunostaining. AR protein levels were measured in rat testes after hormonal treatments at three developmental stages: on days 21, 35, and 90 postpartum. At each age, five groups of animals were treated for 4 days with: 1) vehicle; 2) LHRH antagonist (NalGlu, 0.3 mg/kg BW.day) to suppress endogenous levels of androgen that accompany inhibition of LH and FSH secretion; 3) NalGlu + LH (0.2 mg/kg BW.day); 4) NalGlu + testosterone (T, at 7.5 mg/kg BW.day); and 5) NalGlu + MENT (a potent synthetic androgen, 7 alpha-methyl-19-nortestosterone, 0.7 mg/kg BW.day). AR protein was visualized by immunohistochemistry and measured by computer-assisted image analysis in Leydig and Sertoli cells using frozen sections of tests. After NalGlu treatment, AR levels in Leydig cells declined sharply to 42% and 31% of vehicle control (P < 0.01) in the 21 and 35 days postpartum age groups, respectively, but in 90-day-old rats there was no change. AR levels were partially maintained by exogenous LH, and completely maintained by exogenous androgen treatments in Leydig cells from 21- and 35-day-old rats, whereas in Leydig cells from 90-day-old rats, AR levels were unaffected in all treatment groups. In contrast, after NalGlu treatment, the AR concentration in Sertoli cells from 90-day-old rats were reduced to 32% of control (P < 0.01). Moreover, in Sertoli cells from 90-day-old rats, AR levels were partially maintained by LH and completely maintained by androgens. A similar trend was observed on day 35. On day 21, however, AR levels in immature Sertoli cells were unaffected in all treatment groups. These results indicate that androgen maximally stimulates AR levels in immature Leydig cells but is without significant effect in adult Leydig cells. In contrast, AR levels in Sertoli cells are more sensitive to androgen regulation in adult compared with immature animals. These findings indicate that there are distinct mechanisms controlling AR concentrations in Leydig and Sertoli cells during the development of the testis.

Steroid hormones from the testes promote attraction to estrous females and facilitate copulation in the male Syrian hamster. We compared the ability of testosterone (T) and MENT, a potent synthetic androgen that does not undergo 5 alpha-reduction, to maintain sexual behavior in castrated males. Steroid treatment was initiated immediately after castration at three levels by means of Alzet osmotic pumps in sexually experienced adult male hamsters. Daily doses were 5, 25, or 100 micrograms T and 1, 5, or 25 micrograms MENT (n = 5/group). Additional castrated males (n = 5) remained untreated. Sexual behavior was recorded during two 10-min tests before, and at 2, 4, 6, and 8 weeks after orchidectomy. MENT and T maintained equivalent levels of behavior at each corresponding dose of androgen (high, medium, or low). The low dose of T or MENT failed to sustain mating behavior. Eight weeks after castration, males receiving the high and medium doses of androgens continued to express intromissions and ejaculations at gonadally intact levels. However, only males receiving the high dose showed anogenital investigation at the same level as intact males. From these data, we conclude that MENT sustains mating behavior in the male hamster, and that chemoinvestigatory behavior requires higher levels of androgens than those necessary for copulation.

Tamoxifen is a non-steroidal antiestrogen that possesess antagonistic as well a agonistic properties, while RU39411 is an antiestrogen that is known to possess only antagonistic properties. These steroid antagonists were administered orally to rats, with or without mifepristone, an antiprogestational agent, prior to implantation on Day 2 of pregnancy. The status of pregnancy was assessed on Day 14. Low doses of tamoxifen reduced litter size and weight and inducing embryonic absorption in some animals; a dose of 0.25 mg/Kg prevented pregnancy in all animals. RU39411 also had a dose-dependent effect on pregnancy, but a higher dose (0.5 mg/Kg) was required to achieve the same degree of pregnancy prevention. Addition of mifepristone to both antiestrogens had a synergistic effect on reducing litter size and weight. To determine the mechanism by which antiestrogens terminate pregnancy in rats, oviducts and uteri of treated rats were examined for the presence of embryos on Day 3 and 4 of pregnancy, times when most embryos would be expected to be in the oviducts. Most of the embryos of the treated animals were found in the oviducts on Day 3 of pregnancy. By Day 4, only a few embryos were still present in oviducts. These observations suggest acceleration of embryo transport by Day 4 of pregnancy. There was no accumulation of embryos in the uterus. Since acceleration of embryo transport through the reproductive tract in rat is induced by low doses of estrogens, it is likely that the agonistic action of tamoxifen is responsible for pregnancy prevention in these experiments. The fact that RU39411 also prevents pregnancy by a similar mechanism suggests that this estrogen antagonist also has some estrogen agonist effects on the oviduct. The fact that both antiestrogens also affected embryo weight could suggest the action of the antihormones on other mechanisms controlling embryo development.

Behavioral and endocrine effects of a synthetic androgen, 7 alpha-methyl-19-nortestosterone (MENT), which is not 5 alpha-reduced to dihydrotestosterone, were compared to those of testosterone in two inbred strains of male mice, C57BL/6J and DBA/2J, in two experiments. In the first experiment, seminal vesicle (SV) weights, kidney weights, and circulating steroid levels were examined in castrated mice treated with three doses of testosterone (3.125, 12.5, or 50 micrograms/day) or four doses of MENT (1, 4, 16, or 64 micrograms/day) for 2 weeks to determine the optimal replacement levels of the two androgens for behavioral studies. Both testosterone and MENT dose-dependently increased the SV weights that were greatly reduced, in both strains, by castration. MENT was more effective than testosterone in increasing SV weights, fully restoring them to intact levels in both strains, at the dose of 4 micrograms/day. At the dose of 12.5 micrograms/day, testosterone restored the SV weights completely in C57BL/6J and up to 80% in DBA/2J mice. DBA/2J mice were more sensitive than C57BL/6J mice to both androgens, as measured by kidney weights, although circulating levels of either steroid were very similar between the two strains of mice. In the second experiment, we investigated the effects of testosterone (12.5 micrograms/day) and MENT (4 micrograms/day) on sexual and aggressive behaviors. In each strain, MENT-treated and testosterone-treated mice showed similar numbers of mounts or intromissions. MENT was equally effective as testosterone to fully (C57BL/6J) or partially (DBA/2J) restore sexual behaviors as well as the SV weights to the intact levels. In contrast, MENT-treated mice of both strains were much less aggressive than testosterone-treated mice. In both C57BL/6J and DBA/2J mice, testosterone fully restored aggression to the intact levels as measured by aggression latency, number of aggressive bouts, and duration of aggression, whereas aggressive behaviors of the MENT-treated groups were not different from those of the castrated control groups. These results suggest that MENT can restore both male sexual behaviors and reproductive organ weights as effectively as testosterone, at one-third of the testosterone dose, without stimulating male aggressive behaviors.

RU486, an antiprogestational agent, and anordiol (dihydroxylated metabolite of anordrin) which has an estrogenic and antiestrogenic activity, are known to inhibit fertility. These agents were administered orally, alone or together, to rats prior to implantation, on Day 2 of pregnancy. Control animals were fed with the vehicle only. The effectiveness of the agents in terminating pregnancy in female rats was determined on Day 14 of pregnancy. Anordiol presented a dose-dependent effect on abolishing pregnancy, being 100% effective at 2.5 mg/Kg and non-effective at 0.6 mg/Kg. RU486 did not prevent pregnancy even at a dose of 4 mg/Kg. Doses of RU486 and anordiol that were ineffective when administered alone, prevented pregnancy in 70% of the rats when these agents were given together. To determine the mechanism by which these drugs prevent pregnancy, oviducts and uteri of rats were examined for presence of embryos on Day 3 of pregnancy. Only 29% of embryos were recovered from the oviducts of rats treated with 2.5 mg/Kg anordiol (compared to 89% in control group) plus an additional 9% from the uteri. In combination, anordiol and RU486 had a synergistic effect on embryo transport in the rats' reproductive tract, without any apparent accumulation in the uterus. These results led us to conclude that the pregnancy preventing action of anordiol plus RU486 is mostly due to accelerated transport of the embryos in the reproductive tract prior to implantation.

Testin I and testin II are the two molecular variants of testin that are synthesized and secreted by Sertoli cells in vitro. NTerminal and partial internal amino acid sequence analysis of testin I and testin II reveals that these molecules are identical with the exception that testin II has three extra N-terminal amino acids of TAP compared to testin I. Studies using immunohistochemistry suggested that testin is a component of the specialized junctional complexes in the seminiferous epithelium and other tissues. Immunoreactive testin is localized not only at Sertoli-Sertoli and Sertoli-germ cell junctions, but also at sites of similar junctions in the liver, epididymis, kidney, and intestine. Other physiological studies have shown that the secretion of testin is tightly coupled to the presence of germ cells. In view of its possible role in germ cell development and its unique localization in the cell junction, the purpose of the present study was to determine the structure of testin by sequencing its full-length cDNA. Two synthetic degenerate oligonucleotides based on the N-terminal and an internal amino acid sequence were used for polymerase chain reaction (PCR) to obtain a 289-bp cDNA fragment. This PCR product was subsequently used to isolate a 1371-bp cDNA from a cDNA expression library constructed from Sertoli cell poly(A) RNA. This cDNA coded for a 333 amino acid peptide that starts with an ATG initiation codon from the 5' end and ends with a TGA termination codon located 245 nucleotides before the polyadenylation site. The deduced amino acid sequence indicates that testin contains a 16 amino acid signal peptide with two possible cleavage sites that yield 314 and 317 amino acids for testin I and testin II with calculated molecular weights of 36 029 and 36 299, respectively. Comparison of the entire coding region of testin with existing sequences at Genbank, EMBL, and Protein Identification Resource indicates that testin shares 58%, 57.4%, and 61% identity with rat, mouse, and human cathepsin L at the amino acid level, respectively. The positions of all of the 7 Cys residues and 8 of the 10 Trp residues in testin are conserved with respect to those present in cathepsin L. It is noted that Cys-122 in the predicted active site of cathepsin L was replaced with Ser-122 in testin. In view of the striking primary sequence homology between testin and cathepsin L, we assayed the proteolytic activity of testin using conditions known to activate cathepsin L. Results of these studies showed that no cathepsin L-like protease activity was detected. Northern blot analysis revealed that the testin mRNA was detectable in Sertoli cells, testis, and ovary, but not in brain, liver, kidney, spleen, intestine, epididymis, or uterus from either male or female adult rats. However, hot-nested reverse transcriptase (RT)-PCR yielded a distinctive band with the expected size not only from the RNA of Sertoli cells, testis, and ovary, but also from epididymis, kidney, and intestine. However, the expression of the testin mRNA transcript in the gonads is several orders of magnitude higher than in other tissues, suggesting that its expression is correlated with the rapid tissue remodeling in the gonads during germ cell and follicle development. In conclusion, testin is a newly identified component of cell junction whose mRNA is predominantly expressed in the gonads.

Testin is an authentic Sertoli cell secretory protein consisting of two molecular variants designated testin I (M(r) 35 000) and testin II (M(r) 37 000). N-Terminal amino acid sequence analysis revealed that testin I is identical to testin II except that testin II has three extra N-terminal amino acids of threonine-alanine-proline (TAP). Earlier studies by immunoflorescence microscopy have shown that testin is detected in the seminiferous epithelium consistent with localization in the junctions between Sertoli cells as well as Sertoli-germ cells, and that it appears to be a component of junctional complexes in the testis. In the present study, we have examined the localization of testin in different stages of the spermatogenic cycle of the adult rat testis when germ cells migrate from the basal portion of the seminiferous epithelium to the tubular lumen. In stages I-IV, testin was localized mainly in the basement laminae in the junctional complexes between adjacent Sertoli cells as well as between Sertoli cells, spermatogonia, and pachytene spermatocytes. When elongated spermatids were embedded into the seminiferous epithelium in stage VII of the cycle, testin was detected predominantly on the concave side of the elongated spermatids, but relatively few testin reaction products were seen in the round spermatids. In the beginning of stage VIII of the spermatogenic cycle, intense testin immunoreactive substances were detected around the heads of the elongated spermatids; these substances were virtually undetectable in late stage VIII after the release of the mature sperm into the tubular lumen, suggesting that testin may be a novel marker to divide stage VIII into stages VIIIa and VIIIb.(ABSTRACT TRUNCATED AT 250 WORDS)

Over the last few years, several reviews have focussed on various but limited aspects of antiprogestins in general and RU 486 in particular.lm4 The present review attempts a comprehensive evaluation of the physiology and current clinical uses of RU 486. Progesterone plays a critical role in mammalian reproduction in that it is essential for the initiation and maintenance of pregnancy. Following the discovery of the progesterone receptor,i it was recognized that a progesterone receptor antagonist would be a significant advance in contraceptive technology if it could induce menstruation when used in the luteal phase (once-a-month pill), prevent implantation (morning after pill), and promote abortion in early pregnancy. Furthermore, since progesterone facilitates the action of estradiol in inducing the LH surge prior to ovulation,” a contraceptive action of a progesterone antagonist was also contemplated. The search for such an antiprogesterone extended over more than a decade. The eventual discovery was fortuitous since the scientific team was in fact looking for a glucocorticoid antagonist. (For the interesting story behind the early studies, see reference 1.) In 1981, Philibert and coworkers’ from Roussel-Uclaf reported the first highly effective progesterone antagonist designated RU 38486, subsequently abbreviated to RU 486

The postnatal differentiation of rat Leydig cells may be subdivided into three stages based on morphology and steroid production. The purpose of this study was to clarify the developmental mechanisms underlying increased testosterone production by measuring steady state levels of the mRNAs for three steroidogenic enzymes in isolated Leydig cells at each stage of differentiation. These include Leydig cell progenitors on day 21, immature Leydig cells on day 35, and adult Leydig cells on day 90. The steroidogenic enzymes were 1) cholesterol side-chain cleavage enzyme (CSCC), 2) 17 alpha-hydroxylase (P450-17 alpha), and 3) 3 alpha-hydroxysteroid dehydrogenase (3 alpha HSD). We report that levels of CSCC and P450-17 alpha mRNAs increase, whereas 3 alpha HSD mRNA levels decline during the course of Leydig cell differentiation. The levels of 3 alpha HSD mRNA were high in progenitor Leydig cells that appeared to contain little smooth endoplasmic reticulum and decreased in cells as smooth endoplasmic reticulum developed and other enzyme mRNAs increased. These observations suggest that the factors that regulate 3 alpha HSD mRNA levels are startlingly different from those that regulate the mRNA levels of CSCC and P450-17 alpha. We conclude that the progressive increase in the capacity of differentiating Leydig cells to produce testosterone can be explained in part by an increase in the activity of enzymes that synthesize testosterone (CSCC and P450-17 alpha) and a decrease in the activity of an enzyme that metabolizes testosterone and its precursors (3 alpha HSD).

The synthetic steroid 7 alpha-methyl-19-nortestosterone (MENT) binds with high affinity to the androgen receptor and exerts biological effects at some peripheral target tissues with a potency greater than that of naturally occurring androgens. In vivo, MENT does not undergo enzymatic 5 alpha-reduction and as a consequence, its biologic action on prostate and other organs of the male reproductive tract is not amplified as is that of testosterone (T). Thus, in castrated rats, a dose of MENT that will maintain normal muscle mass and gonadotropin levels will not maintain normal prostate and seminal vesicle weights. To investigate the ability of MENT to restore male sexual behavior in castrated rats, varying doses of MENT acetate were administered for 4 wk by use of s.c. mini-osmotic pumps. Animals treated with T acetate (200 micrograms/day) and nontreated intact animals served as positive controls, while a group of animals receiving vehicle alone were the negative controls. Steroid acetates are rapidly converted to T and MENT in blood. Appropriate steroid delivery was assessed by measurement of serum androgen concentrations. Male behavioral parameters were recorded twice per week. At the end of treatment, the weights of sex accessory organs were also recorded. The administration of MENT acetate at daily doses of 100 micrograms and 10 micrograms induced full copulatory behavior in a manner similar to that observed with doses of 200 micrograms T acetate.(ABSTRACT TRUNCATED AT 250 WORDS)

In the rat reproductive tract, sulfated glycoprotein 2 (SGP-2) is present in the ventral prostate, seminal vesicle, testis, and epididymis. In the ventral prostate, SGP-2 is associated with the process of programmed cell death, while in the testis and epididymis a role for SGP-2 in sperm maturation has been proposed. Available information suggests that there are both inter- and intra-organ variations in SGP-2 localization, molecular forms, and response to androgen ablation. In the present study, localization of SGP-2 within the ventral prostate, seminal vesicle, and epididymis was compared by immunohistochemistry. In the ventral prostate of intact rats, immunoreactive SGP-2 was confined to a discrete population of epithelial cells lining the proximal ducts. Epithelial cells in other regions of the ventral prostate did not stain for SGP-2. A similar staining pattern was observed for the seminal vesicle; a small population of SGP-2-expressing epithelial cells was found in epithelium that did not stain for SGP-2. The epididymis also demonstrated a non-uniform staining pattern. The caput displayed strong immunoperoxidase reaction over the apical membrane and stereocilia of all principal cells. Principal cells also showed variable degrees of cytoplasmic staining ranging from weak to strongly positive. The corpus and cauda showed a similar staining pattern. After castration, all epithelial cells in the ventral prostate and seminal vesicle became intensely positive for SGP-2 staining. In the caput and cauda epididymis there was an increase in the number of principal cells demonstrating strong intracellular staining for SGP-2. These results suggest that as observed previously in the regressing ventral prostate, increased intracellular SGP-2 staining may also be associated with the regressing epididymis and seminal vesicle. Differences in molecular forms of SGP-2 were investigated by two-dimensional Western and lectin blots. Molecular forms of SGP-2 differed between testis and epididymis but were similar between ventral prostate and seminal vesicle. Prostate and seminal vesicle forms of SGP-2 differed from those of both testis and epididymis. Analysis of terminal carbohydrate present on the various SGP-2 molecular forms also confirmed the existence of heterogeneity. These results demonstrate the presence of multiple molecular forms of SGP-2 in various organs of the male reproductive tract in rats and suggest a possible variation in functional activity and/or half-life of SGP-2 in these organs.

In view of the unexpected ability of anordrin to synergize with RU 486 in terminating pregnancy, it was pertinent to examine the actions of the dihydroxylated metabolite of anordrin, anordiol, alone and in combination with RU 486. Does of RU 486 (1 mg/kg/day) and anordiol (0.6 mg/kg/day) that were ineffective when given alone terminated pregnancy with complete resorption of embryos when administered together. A smaller dose of anordiol than anordrin is required to achieve this synergistic effect with RU 486. This anordrin metabolite increased uterine weight in the ovariectomized rat similar to estradiol. The estrogenicity of anordiol in the uterine weight assay was about 1/120 of that of estradiol. Anordiol does not exert antiestrogenic activity in the uterine weight assay when administered at doses that terminate pregnancy. Administration of anordiol at doses that do not terminate pregnancy resulted in a significant suppression of serum progesterone concentrations during the period of medication; these observations suggest that anordiol has an inhibitory effect on progesterone biosynthesis. When the same dose of anordiol was given concomitantly with sufficient RU 486 (e.g., 1 mg/kg/day) to terminate pregnancy, the progesterone levels were reduced to low levels throughout the experiment. These observations support the postulate that the actions of anordrin are mediated by its metabolite, anordiol. The administration of anordiol plus RU 486 results in a more dramatic change in the functional progesterone:estradiol ratio than when either agent is administered alone.

The effectiveness of a combination of RU 486, an antiprogestin, and epostane, a 3β-hydroxysteroid dehydrogenase inhibitor, for termination of pregnancy in female rats was determined. Epostane administered at doses ranging from 8 to 48 mg/Kg/day on days 7, 8, and 9 of pregnancy did not influence the progress of the pregnancy. RU 486 in doses of 1, 2, and 4 mg/Kg/day terminated pregnancy in 0, 40, and 100% of rats, respectively. The combination of epostane (48 mg/Kg/day) plus RU 486 (1 and 2 mg/Kg/day) terminated pregnancy with complete resorption of fetuses in all treated animals. Unexpectedly, the drug combination that terminated pregnancy used a dose of epostane that was not highly effective in lowering serum progesterone levels when epostane was administered alone. Thus, in the rat, the synergistic action of epostane plus RU 486 may not be dependent upon reduced progesterone secretion plus receptor blockage as anticipated. The present results suggest that the combination of epostane plus RU 486 is effective for pregnancy termination.

aO 2 -Macroglobulin and clusterin are two putative Sertoli cell secretory products; however, the regulator(s) modulating their secretion by Sertoli cells is not known. Recent studies from this laboratory have shown that the testicular a2-macroglobulin, unlike its liver homologue, is not an acute-phase reactant and its concentration is not affected by acute inflammation. We sought to determine whether FSH, testosterone, and other biomolecules would affect the secretion of oi,-macroglobulin and clusterin by Sertoli cells as well as whether peritubular myoid cells would affect the secretion of these proteins by Sertoli cells. It was noted that Sertoli cells cultured in vitro secreted increasing amounts of oa 2 -macroglobulin and clusterin as a function of time. FSH (50-1000 ng/ml) and testosterone (10-'-10 - 5 M) had no apparent effect on the secretion of a 2 -macroglobulin and clusterin by Sertoli cells. Addition of interleukin-6 to Sertoli cell-enriched cultures, in doses known to stimulate u,-macroglobulin secretion by hepatocytes, did not affect the ot,-macroglobulin secretion. However, dexamethasone at 10-7-10 - 5 M stimulated t,-macroglobulin secretion by Sertoli cells dose-dependently while the addition of interleukin-6 had no synergistic effect on dexamethasone-stimulated at2-macroglobulin secretion. These findings suggest that the synthesis and/or secretion of ot2-macroglobulin by Sertoli cells is regulated by a mechanism distinct from that of the liver. Peritubular cells cultured alone did not produce any measurable amounts of either aot-macroglobulin or clusterin; however, co-cultures of peritubular cells with Sertoli cells induced a significant increase in i2,-macroglobulin and clusterin secretion by Sertoli cells. Addition of testosterone to the Sertoli cell-peritubular cell co-culture system did not affect the levels of 2,-macroglobulin and clusterin. In summary, FSH and testosterone, at doses known to affect Sertoli cell function, did not affect the secretion of aot-macroglobulin and clusterin; however, peritubular cells modulate the secretion of both a 2-macroglobulin and clusterin by Sertoli cells.

Clusterin, a protein associated with cell death, has been suggested as a marker of renal injury. Correlation of clusterin gene expression with changes in renal function and quantitative measurement of clusterin protein levels after ureteral obstruction have not been previously reported. With unilateral ureteral obstruction in rabbits as the experimental model, the time course of alterations in renal function, clusterin mRNA accumulation, and concentrations of clusterin protein in serum, urine, and renal tissue were investigated. RBF, GFR, and renal concentrating ability (percent sodium reabsorption and urine osmolarity) all decreased (P0.05) in the obstructed kidney from control values within 1 day of ureteral obstruction. Clusterin mRNA levels started to rise in the ipsilateral kidney within 12 h of ureteral obstruction and increased up to 10-fold above control levels after 3 days of obstruction. Hybridization histochemistry showed that clusterin mRNA was initially detectable in collecting ducts and distal tubules within 12 h of ureteral obstruction. After 7 days of obstruction, increased accumulation of clusterin mRNA was also detectable in proximal tubular epithelial cells. Clusterin gene expression remained elevated in collecting ducts after 60 days of obstruction. Clusterin expression in the contralateral kidney was increased twofold over control values after 12 h of obstruction. No increase in clusterin mRNA accumulation was detectable after 24 h in the contralateral kidney. Total clusterin protein in the obstructed kidney increased from 0.59 +/- 0.66 (mean +/- 1 SD) to 2.5 +/- 1.3 micrograms after 7 days of ureteral obstruction (P0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

The aim of this study was to determine the bidirectional release of Immunoreactive inhibin-a (irINH.u) by different testicular compartments in the adult ram and to assess the effects of FSH on the distribution of inhibin in the testis. Immunoreactive INH-a was measured by RIA in fluid samples collected concurrently from the three testicular compartments-the seminiferous tubules, the interstitium, and the vascular system-through catheters inserted surgically into the rete testis, testicular lymphatic duct system, and spermatic veins, respectively, Overall, the concentration of iriNHix in rete testis fluid was 25 times the level in testicular lymph and over 500 times the concentration in peripheral blood. The pattern of irINH-ot concentration in rete testis fluid was inverselyrelatedto that in testicular lymph, but i,v, administration of FSH had a decoupling effect on this relationship by depressing inhibin concentration in testicular lymph without affecting inhibln levels in rete testis fluid. Nevertheless, increased flow of testicular lymph more than compensated for the transient fall in irINH-u concentration so that, overall, the total output of Inhibin via the testicular lymphatic duct system (and the vascular system) increased significantly. No persistent or significant changes were observed in the flow rate of rete testis fluid or concentration of irINH-a In the fluid after administration of FSH. The time frame for the response of the testis to FSH is indicative of the involvement of a mediator. Electrophoretic analysis of serially collected testicular lymph samples consistently revealed an F5H-induced release of a series of proteins in the M, range of 30 000-32 000. These results point to an FSH-induced increase in vascular permeability as a major mechanism of inhibIn release by the outer compartment of the seminiferous epitheium.

UNLABELLED Testin I and Testin II were originally identified as Sertoli cell products with similar NH2-terminal amino acid sequences. Secretion of testins is stimulated by testosterone in Sertoli cell-enriched cultures. By contrast the secretion of testins from intact seminiferous tubules appears to be inversely related to germ cell number. In the present study testin antiserum that recognized both Testin I and Testin II ("testin") was used to localize these proteins in tissue secretions by immunofluorescence. Testin was localized at the base of the seminiferous epithelium at Sertoli-Sertoli junctions. Fluorescence also appeared to be located at the sites of interaction between spermatoids and Sertoli cells. A punctate pattern of fluorescence was also present in the cytoplasm of Leydig cells; without electron microscopic studies it was not possible to determine which structures the antibodies bound to in these cells. In the epididymis the reaction product was localized at the apices of the epithelial cells adjacent to the lumen at the sites of known junctional complexes. A variety of positive and negative controls indicated that staining was specific for testins. CONCLUSIONS This is the first study to associate testins with junctional complexes. Relative to other junctional proteins, testins are unusual because of their small size and because they are secreted proteins.

Based on the premise that testosterone, but not 7 alpha-methyl-androgens, is reduced at the 5 alpha-position in the prostate and seminal vesicles, the differential bioactivities of these androgens were investigated in castrated rats. The ability of 7 alpha-methyl-19-nortestosterone acetate (MENT) to increase the weights of ventral prostate and seminal vesicles of castrated rats was four times higher than that of testosterone, while its effect on the weights of bulbocavernosus plus levator ani muscles (muscle), was 10 times that of testosterone. MENT was also approximately 12 times more potent than testosterone in the suppression of serum gonadotropin levels. A dose of testosterone that maintains serum gonadotropin levels and muscle mass also maintains prostate and seminal vesicle weights in castrated rats. By contrast, a dose of MENT that maintains muscle and gonadotropins does not maintain prostate and seminal vesicles. The action of other 7 alpha-methylated androgens were similar to that of MENT. The importance of 5 alpha reductase in the differential action of testosterone and MENT on prostate was confirmed by using a 5 alpha-reductase inhibitor. The activity of testosterone was significantly suppressed in the ventral prostate and seminal vesicles but not on muscle by the 5 alpha-reductase inhibitor (N,N-diethyl-3-oxo-4-aza-5 alpha-androst-1-ene-17 beta-carboxamide). The enzyme inhibitor, however, had no influence on the activity of MENT on either tissue. In contrast, cyproterone acetate, an antiandrogen that competitively binds to the androgen receptors, inhibited the action of MENT and of testosterone on the prostate as well as on the muscle. In conclusion, these observations show that 7 alpha-methylated androgens can maintain muscle mass and normal gonadotropin levels in androgen deficient rats without hyperstimulating the prostate. These findings suggest that 7 alpha-methylated androgens may offer some health benefits to men who require androgen treatment.

Measurements of tissue immunoassayable clusterin, a protein associated with programmed cell death and tissue reorganization, were performed in rats treated with nephrotoxic doses of gentamicin sulfate. Adult Lewis rats were treated with 100 mg/kg/day of gentamicin sulfate for 12 days. Urine, serum, and tissue levels of clusterin protein were measured, as were urinary N-acetyl beta-glucosaminidase (NAG) and serum creatinine levels. Induction of renal injury by gentamicin was detectable within 4 days by increased levels of urinary N-acetyl beta-glucosaminidase (from 280 +/- 66 (mean +/- SD) to 910 +/- 210 nmol/mg creatinine), and within 9 days of initiating gentamicin treatment by increased serum creatinine (from 0.5 +/- 0.1 to 1.2 +/- 0.4 mg/dl). Paralleling these changes, renal, urinary, and serum levels of clusterin increased 10-, 116-, and 3-fold (P less than 0.05). Treatment with gentamicin sulfate did not increase clusterin levels in the seminal vesicle, ventral prostate, testis, or epididymis. The measurement of urinary or serum clusterin may play a role in the early detection of renal injury.

The study was done to assess the clinical performance and in vivo steroid release rate of 3-keto-desogestrel subdermal implants designed to deliver 5 different doses of the progestin. Volunteers were healthy women of proven fertility who provided blood samples at scheduled intervals during treatment. No pregnancy occurred in 514 woman-months in users of implants delivering 30 and 40 micrograms per day of 3-keto-desogestrel. Three pregnancies, one ectopic, were observed in 109 woman-months recorded with implants delivering 20 micrograms per day or less. Ovulation was inhibited, as judged by depressed progesterone levels, in 57 of 59 (97%) blood samplings in women whose 3-keto-desogestrel plasma levels were greater than 0.28 nmol/L and in 39 of 75 (52%) of cases with lower levels. Users of 4 cm implants manufactured by The Population Council, New York, showed mean levels above 0.28 nmol/L until 18 months of use. Levels achieved with 4.4 cm implants manufactured by Organon, Oss, Holland, were less consistent. No changes were observed in the plasma lipoprotein pattern or clinical chemistry during treatment. The main complaint was the occurrence of bleeding irregularities, particularly with the lower doses. Ovarian cysts found during pelvic examination in 11 (22%) subjects disappeared spontaneously within 7-90 days. 3-keto-desogestrel implants releasing around 40 ug/day and providing plasma levels around 0.28 nmol/L afford efficient contraceptive protection.

Nucleotide sequence analysis of the complimentary DNAs (cDNA) and N-terminal amino acid sequence analysis have shown that clusterin is equivalent to sulfated glycoprotein-2 (SGP-2), testosterone-repressed prostate protein-2 (TRPP-2), and androgen-repressed protein (ARP) in the rat, as well as serum/seminal plasma protein, SP-40,40, in the human. In view of its widespread presence in various species, a specific RIA was established to quantify the tissue distribution of this protein. Rat clusterin is present in almost all organ tissues examined, including testis, epididymis, serum, liver, prostate, seminal vesicles, and uterus. Displacement curves generated using cytosols prepared from these organs were parallel to those obtained using purified rat clusterin and crude Sertoli cell-enriched culture medium. Immunoreactive clusterin was also visualized in these organ extracts by immunoblots. Studies on the tissue distribution of immunoreactive clusterin using RIA revealed that the concentration of clusterin in the epididymis of adult rats was 6- and 10-fold higher than that in the serum and testis, respectively and is 50- to 100-fold higher in the liver, spleen, kidney, brain, ventral prostate, seminal vesicles, and uterus. A study of the distribution of clusterin in various compartments of the epididymis indicated its concentration in the caput epididymis was almost 3-fold higher than that in the corpus and cauda epididymis. After orchiectomy, the concentrations of clusterin in the ventral prostate and seminal vesicles increased as much as 100- and 10-fold and peaked at day 4 after surgery, respectively; daily injection of dihydrotestosterone (DHT) beginning at day 3 after orchiectomy reduced the concentrations of clusterin and restored them to a normal level. A different pattern was noted in the epididymis after orchiectomy; the concentration of clusterin in the caput epididymis decreased with time; however, daily injection of DHT beginning at day 3 increased the caput epididymal clusterin concentration and restored it to a normal level. The concentration of clusterin was not altered in the corpus or cauda epididymis after castration and/or DHT administration. Also, the serum and liver clusterin levels did not change with time after orchiectomy. These observations suggest that clusterin will be a valuable marker to monitor the diverse effects of androgen withdrawal in the male reproductive tract. We conclude that clusterin may be a multifunctional protein in view of its broad tissue distribution and association with numerous physiological and pathological conditions.

Summary We examined the hypothesis that induction of reversible testicular atrophy, subsequent to withdrawal of gonadotrophin support, would alleviate the testicular toxicity of the anti-cancer drug procarbazine. In rats, severe but reversible testicular atrophy and suppression of spermatogenesis were induced 56 days after the subcutaneous insertion of a silastic implant containing oestradiol-17β. The effect of this treatment upon the testicular toxicity of four weekly doses of procarbazine (200 mg kg-1) was examined 56 days after the termination of procarbazine/oestrogen treatment. At this time the testicular endocrine and sper-matogenic functions were close to normal in rats which has received only oestra-diol-17β. Procarbazine produced severe testicular atrophy which was associated with azoospermia and destruction of the germinal epithelium. Serum LH and FSH concentrations were raised and were associated with low serum concentrations of both testosterone and androgen-binding protein. The combination of procarbazine with the oestrogen treatment did not change any of the testicular toxicity and in some cases it appeared to be exacerbated. In contrast to these experiments other studies have indicated that the testis can be protected if spermatogenesis is revers-ibly suppressed by other agents which are also active via the pituitary endocrine system. The data would therefore suggest that protection is achieved either by some testicular change other than withdrawl of pituitary gonadotrophin support or that oestradiol-17β has additional activity which is permissive for the development of the testicular toxicity of procarbazine.

The suppressive effect of the gonadotropin-releasing hormone (GnRH) antagonist Nal-Glu ([Ac-D2Nal1, D4ClPhe2, D3Pal3, Arg5, D-4-p-methoxybenzoyl-2-aminobutyric acid6, DAla10]-GnRH), injected intramuscularly with 5 mg, was studied in six men. Testosterone decreased by 87 +/- 2.3%, whereas the mean drops were 50 +/- 10%, 43 +/- 6.6%, and 39 +/- 5.6% for radioimmunoassayable luteinizing hormone (LH), follicle-stimulating hormone, and free alpha-subunit, respectively (mean +/- SEM). Immunological characteristics of plasma LH were modified during the inhibition and recovery phases as evidenced by comparison between polyclonal and monoclonal assays. In two additional subjects sampled every 10 minutes, both LH and alpha-subunit pulses were suppressed by NalGlu injection and restored by pulsatile GnRH infusion. However, a nonpulsatile and possibly non-GnRH-dependent alpha-subunit secretion was maintained after NalGlu administration.

The efficacy and specificity of human recombinant inhibin alpha-subunit as an immunogen were determined by studying its effect on the ovulation rate and serum gonadotropin levels in ewes. Beginning on July 12 (day 1 of the experiment), 2-yr-old Rambouillet ewes were immunized four times at intervals of 20, 30, and 30 days, respectively, by im injections of the alpha-subunit each time with 100 micrograms. Blood was collected from the jugular vein as required before and after ovulation. Ovulation rates were determined, beginning on day 87, by laparotomy before estrous synchronization by means of vaginal progestagen sponges. After synchronization, ewes were examined again. The ovulation rate before estrous synchronization was over 4 times higher in immunized than control ewes [8.7 +/- (+/- SE) 1.9 vs. 2.0 +/- 0.0]. After estrous synchronization, ovulation rates in the immunized and control ewes were 6.3 +/- 2.4 and 1.3 +/- 0.3, respectively; this increase was accompanied by higher pre- and postovulatory FSH levels, but there was no change in the pattern of circulating LH. Furthermore, the postovulatory FSH surge persisted for longer periods of time in the immunized than control animals. These results demonstrate the potency of the human recombinant alpha-subunit as an immunogen and illustrate its specificity in suppressing the effects of endogenous inhibin. The results also point to inhibin as an important regulatory factor in controlling the ovulation rate by modulating both pre- and postovulatory FSH levels.

The mechanism by which the seminiferous epithelium limits the damaging effects of proteases that are released from degenerating late spermatids does not depend upon protease inhibitors in the systemic circulation since these proteins are excluded from the seminiferous tubule by the blood-testis barrier. The purpose of this study was to identify the major protease inhibitor of the testis and determine its cellular origin. Sertoli cells, the major epithelial components of the seminiferous epithelium, release a protease inhibitor, testicular {alpha}{sub 2}-macroglobulin, in vitro. Immunoprecipitation using ({sup 35}S)methionine and a monospecific polyclonal antibody prepared against purified testicular {alpha}{sub 2}-macroglobulin establishes that this protein is actively synthesized and secreted by Sertoli cells. Measurements of immunoreactive protease inhibitors in tubular and rete testis fluids collected by micropuncture suggest that {alpha}{sub 2}-macroglubulin rather than {alpha}{sub 1}-antitrypsin is the major protease inhibitor in the seminiferous tubules in vivo. The ability of {alpha}{sub 2}-macroglobulin to inactivate proteases and growth factors such as TGF-{beta} by a common mechanism suggests that this protein may have a dual function in the testis.

Adult Suffolk rams were immunized four times against the human recombinant inhibin alpha-subunit over a period of 80 days. Blood samples were collected at weekly intervals and serum levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone were determined by radioimmunoassay procedures. The results show that season-related elevations of gonadotropin levels in immunized rams was delayed by 1-2 wk and, in these animals, it was more pronounced and extended than in vehicle-treated controls. Peaks of circulating testosterone were higher in control rams than in immunized animals. The capacity of the antisera to bind 125I-labeled inhibin alpha-subunit increased significantly in each immunized animal within 30 days of treatment, even though neutralizing antibodies were detected with a rat pituitary cell culture bioassay in only one of the four immunized rams. Epididymal sperm reserves tended to be greater in immunized than in control animals. These results show that inhibin controls the release of FSH during the breeding season, thereby regulating spermatogenic activity; it may also exert its effect on testicular function by a local effect on Leydig cells, as evidenced by changes in serum testosterone profiles and increased serum LH levels in rams immunized against the inhibin alpha-subunit.

[Ac-D2Nal1,4Cl-DPhe2,D3Pal3,Arg5,DGlu6+ ++ (anisole adduct),DAla10]-GnRH (Nal-Glu) is an antagonist of LHRH and has the potential to be utilized as an antigonadal agent. A study was undertaken to evaluate the toxicological effects of Nal-Glu in rats. Nal-Glu, dissolved in 5% mannitol in water containing 9 ml/liter benzyl alcohol, was administered subcutaneously. In subchronic studies, groups of 12 male and 12 female rats received 0, 50, 250, or 1250 micrograms/kg body weight (BW) Nal-Glu for 90 days and were killed on Day 91. Additional groups of male and female rats were given the high dose of Nal-Glu (1250 micrograms/kg BW) or vehicle for either 30 or 90 days. Their fertility was assessed by mating them with normal animals. Unlike some other LHRH antagonists, Nal-Glu exhibited a low potency for causing in vitro histamine release from rat peritoneal mast cells. Furthermore, in acute in vivo studies, Nal-Glu was less active in the induction of peripheral edema. In the subchronic study, all doses of Nal-Glu were well tolerated and there were no apparent systemic toxic effects. The pharmacological effects of Nal-Glu were quite evident, however. Nal-Glu treatment led to a significantly decreased body weight gain in the males and a significantly increased body weight gain in the females. There was a dose-dependent decrease in weights of gonads and reproductive organs in both the sexes. Some of the hematological and serological parameters were significantly different in Nal-Glu-treated animals. However, most of the values were within the normal range and are considered to be of no toxicological significance. Histopathological evaluations were made in the control and high-dose groups only. In the male, a seminiferous tubular degeneration and atrophy of the interstitial cells was seen. The prostate and seminal vesicles were also atrophied and the epididymides were devoid of spermatozoa. In the females, the ovaries and uteri were atrophic. The injection site of Nal-Glu-treated rats had inflammatory changes indicative of a local irritating action of the drug. All other tissues had normal histomorphology. Both male and female rats became infertile when 1250 micrograms/kg Nal-Glu was administered for 30 days. Normal fertility was restored 8 weeks after cessation of 90-day treatment. It is concluded that repeated administration of Nal-Glu leads to reversible infertility in both male and female rats. Although it was irritating at the site of injection. Nal-Glu had no systemic toxicological effects.

Androgen is essential for maintenance of spermatogenesis in the testis and for maturation of spermatozoa in the epididymis. The effects of androgen are mediated through its receptor (AR), the levels of which are, in turn, regulated by androgen. Previous studies have shown that AR concentrations in Leydig and Sertoli cells are differentially regulated during development. The aim of the present study was to determine if cell-type-specific regulation of AR by androgen occurs in testicular and epididymal cells during adulthood. Adult male rats were treated with the LHRH-antagonist Azaline B (100 g/day) by osmotic pump for 1, 2, 3, 4, or 8 wk to suppress endogenous androgen, with identical numbers of intact control animals at each time period. An androgen replacement group was simultaneously treated with the antagonist and a synthetic androgen, 7 alpha-methyl-19-nortestosterone (MENT), during the final 4 wk of the experiment. Levels of nuclear AR protein in specific cell types were quantified by immunohistochemistry in conjunction with computer-assisted image analysis. Levels of AR in testicular cells declined sharply after treatment with the LHRH antagonist. In Sertoli cells, nuclear AR levels decreased to 8% of control (P0. 01) after 4 wk treatment; and to 12% and 17% of control (P0.01) in Leydig and myoid cells, respectively. Androgen replacement resulted in complete recovery of nuclear AR levels in Sertoli cells (93%, P0.05) but in only partial recovery in myoid (69%, P0. 01) and Leydig cells (56%, P0.01). In the epididymis, tubular epithelial cells and stromal cells differed in their responses to the LHRH antagonist. After 1 wk, nuclear AR levels in caput stromal cells decreased dramatically to 34% of control (P0.01) and in cauda stromal cells to 43% (P0.01). In contrast, the decline of AR levels in epididymal epithelial cells was not as dramatic as that in stromal cells. After 1 wk, the decline in the caput and cauda was to 87% and 76% of control, respectively. After 8 wk, nuclear AR levels in stromal cells further declined to 1.1% in caput and 1.4% in cauda, whereas in the epithelial cells, a smaller decline in nuclear AR was noted (to 30% in the caput and 45% in the cauda). After androgen replacement with MENT, nuclear AR levels recovered to more than 90% of control in both epididymal cell types. These results indicate that AR levels in the nuclei of adult Sertoli cells depend mainly on the level of androgen, whereas in the adult Leydig and myoid cells, the androgen dependency is more limited. The results also indicate that in the epididymis, stromal cells are more sensitive than epithelial cells to the regulation of AR levels by androgen.

CNNT. There was a good correlation between bioactivity and binding affinity to AR for the 7alpha-substituted androgens compared to T. In contrast, relative to their binding affinity to AR, the androgenic potency of DHT and 19-NT was lower compared to T. The reason for the lower in vivo androgenic activity of 19-NT is attributable to its enzymatic conversion to 5alpha-reduced-19-NT in the prostate. In the case of DHT, the lower bioactivity could be attributed to its faster metabolic clearance rate relative to T. The correlation was further investigated in vitro by co-transfection of rat ARcDNA expression plasmid and a reporter plasmid encoding the chloramphenicol acetyl transferase (CAT) gene driven by an androgen inducible promoter into CV-1 cells. All the androgens led to a dose-dependent increase in the CAT activity. MENT was found to be the most potent followed by DHT, 19-NT, T, and CNNT. The specificity of the androgenic response was confirmed by its inhibition with hydroxyflutamide, an antiandrogen. Thus, there was a good correlation between binding affinity and in vitro bioactivity in the transient transfection assay for the androgens. This suggests that the in vivo bioactivity of androgens could be influenced not only by binding affinity to receptors but also by factors such as absorption, binding to serum proteins and metabolism. However, the high potency of MENT is primarily related to its higher affinity to AR.

7alpha-Methyl-19-nortestosterone (MENT) is a potent synthetic androgen that cannot be converted to dihydrotestosterone. In this study we determined the relative androgenic, antigonadotropic, and anabolic potencies of testosterone vs. MENT in the nonhuman primate M. fascicularis. In castrated monkeys, dose-response relationships were generated for the effects of testosterone and MENT on gonadotropin levels, prostate growth, body weight, and lipid metabolism. In a pilot study, four monkeys were castrated, and magnetic resonance imaging (MRI) was used to document a 50% loss of prostate volume within 8 weeks, verifying that MRI is a reliable means to measure prostate size in this species. Two additional groups of six monkeys each were then castrated and serially administered four graded dosages of testosterone or MENT via osmotic minipumps over 20 weeks. Complete suppression of LH was achieved with a minimum of 0.3 mg/day MENT, compared to 3.0 mg/day testosterone. MENT supported body weight 10 times more potently than did testosterone. Baseline prostate volumes were maintained with 0.1-0.2 mg/day MENT vs. 0.3 mg/day testosterone. Thus, in monkeys, MENT is 10 times more potent than testosterone with regard to the clinically desirable end points of gonadotropin suppression and anabolism, but only twice as potent at stimulating prostate growth. These results suggest that MENT may have a wider therapeutic index than testosterone for human androgen replacement and male contraception.

Mifepristone and a prostaglandin have been used successfully to terminate pregnancy in Europe and China. We report the results of a large U.S. study of mifepristone and misoprostol in women with pregnancies of up to nine weeks' duration.We administered 600 mg of mifepristone and then 400 microg of misoprostol two days later to 2121 women seeking termination of their pregnancies at 17 centers. The women were observed for four hours after the administration of misoprostol and returned on day 15 for final assessment.Two thousand fifteen women completed the final assessment. Among them, pregnancy was terminated in 762 of the 827 women pregnant foror =49 days (92 percent), 563 of the 678 women pregnant for 50 to 56 days (83 percent), and 395 of the 510 women pregnant for 57 to 63 days (77 percent) (P0.001). Termination occurred within 4 hours after the administration of misoprostol in 49 percent of the women and within 24 hours in 75 percent. Failures, defined as cases requiring surgical intervention for medical reasons or because the patient requested it, the abortion was incomplete, or the pregnancy was ongoing, increased with increasing duration of pregnancy. The largest increase was in failures representing ongoing pregnancy, which increased from 1 percent in theor =49-days group to 9 percent in the 57-to-63-days group (P0.001). Abdominal pain, nausea, vomiting, diarrhea, and vaginal bleeding also increased with advancing gestational age. Two percent of the women in theor =49-days group, as compared with 4 percent in each of the other two groups, were hospitalized, underwent surgical interventions, and received intravenous fluids (P=0.008).This mifepristone-misoprostol regimen is effective in terminating pregnancies, especially in women with pregnancies of 49 days' duration or less.The availability of medical abortion in the US and elsewhere could lead to greater access to safer abortion services. This study assessed the capability of mifepristone (600 mg) and misoprostol (400 mcg 2 days later) to terminate pregnancies of up to 63 days' duration. Enrolled were 2121 US women recruited from 17 Planned Parenthood, university hospital, and free-standing abortion clinics. Among the 2015 women who returned for the follow-up visit at day 15, the rates of pregnancy termination were 92% in women with pregnancies of durations of 49 days or under, 83% in the 50-56 days group, and 77% in the 57-63 days group (p 0.001). Abortion occurred within 4 hours of misoprostol administration in 49% of women and within 24 hours in 75%. Termination rates were higher for women with no previous induced abortions. Side effects such as abdominal pain, nausea, vomiting, diarrhea, and vaginal bleeding were frequent and increased with gestational age. 2% of women with pregnancies of 49 days' gestation or less, compared with 4% in both the 50-56 days and 57-63 days groups, were hospitalized, underwent surgical intervention, and received intravenous fluids. The success rate in this study was lower than that reported by other researchers. This may be related, in part, to the lack of experience with medical abortion in the US.

Somatostatin (SRIF) exerts multiple inhibitory actions throughout the body by binding to specific SRIF receptors (sst). In recent years, five subtypes of SRIF receptors (sst1-5) have been cloned. In this study, 35S-labeled complementary RNA probes were used for in situ hybridization to localize the sst1-5 messenger RNAs (mRNAs) in the rat testis and examine the changes in their distribution during the cycle of the seminiferous epithelium. We found that sst 1-3 mRNAs were visualized in rat testes and were mainly localized within the seminiferous tubules. The signal for sst3 mRNA was also found in interstitial cells. sst4 and 5 mRNAs were not detected in rat testes with the method used in this study. In Sertoli cells, the most intense labeling for sst1 and 3 mRNAs was in stages IV-VII of the cycle of the seminiferous epithelium, which coincided with the lowest labeling intensity for sst2. In germ cells, sst1-3 mRNAs showed similar patterns of distribution. In these cells, sst1-3 mRNA was not observed at the early steps of spermatogenesis. Positive signals for sst1-3 mRNAs were first apparent in the pachytene spermatocytes at stage VII and last until stage XII and in the diplotene spermatocyte at stage XIII. Positive signals for sst1-3 were also detected in round spermatids at stages I-VIII. Labeling of spermatids dramatically decreased at stage IX, when these cells began their elongating changes. The presence of three sst in testis suggests that SRIF may play an essential role in testicular function.

Intermediate filaments (IFs) are components of the cytoskeleton. In mammalian Sertoli cell, IFs are formed by vimentin. Previous studies have shown some characteristics of its distribution in Sertoli cells, however, very little is known of its distributional changes during the seminiferous epithelium cycle and during postnatal development.Immunohistochemical and electron microscopic methods were used to determine the distribution of vimentin-type IFs in rat Sertoli cells during the seminiferous epithelium cycle and postnatal development.The distribution of IFs in adult rat Sertoli cell showed distinct cyclic changes during the seminiferous epithelium cycle. At stages I-VI, bundles of IFs extend from the perinuclear region to the supranuclear and apical regions of the Sertoli cell. These apical extensions became shorter at stage VII, and at stages VIII-X IFs were observed only in the perinuclear region. Short apical extensions reappeared at stages XI-XII; and at stages XIII-XIV, they extended again into the apical region. During this cycle, IFs were always closely associated with the heads of elongate spermatids. IFs were also shown to be in close apposition to some specialized structures on the cell membrane, such as the ectoplasmic specialization between adjacent Sertoli cells. During postnatal (p.n.) development, IFs were mainly observed at the basal nuclear region on p.n. day 7. The IFs in the supranuclear or apical regions first appeared at p.n. day 14 and gradually increased during the development. The perinuclear IFs network was fully established by p.n. day 28 and the adult distribution pattern of the IFs was established by p.n. day 42.Vimentin-type IFs in rat Sertoli cells are a delicate endocellular network, which is centered in the perinuclear region and extends to the apical region of the cell. During the seminiferous epithelium cycle, the distribution of IFs changes in a stage-dependent manner and is closely related to the location of the heads of elongate spermatids. During postnatal development, IFs gradually increase in numbers and the main distribution area is transferred from the basal nuclear to the perinuclear and supranuclear regions.

Testosterone and its esters are widely used for androgen replacement therapy. In the prostate, testosterone ins 5 alpha-reduced to dihydrotestosterone (DHT), which leads to an amplification of its stimulatory activity in this and other tissues that have significant 5 alpha-reductase activity. While this amplification is essential during fetal development, it has potentially undesirable consequences during adult life. 7 alpha-Methyl-19-nortestosterone (MENT) is a potent synthetic androgen that does not undergo 5 alpha reduction and is therefore being investigated for long-term clinical use because it is expected to be less stimulatory to the prostate. Since we anticipate using MENT acetate (MENT Ac) rather than MENT as the form of this androgen in humans, the bioavailability of MENT following the administration of MENT and MENT Ac was investigated in cynomolgus monkeys. Equimolar concentrations of MENT or MENT Ac were administered as a continuous subcutaneous infusion via Alzet osmotic pumps. Serum MENT levels were measured by radioimmunoassay (RIA) in blood samples collected daily for 4 days during steady state. The serum MENT levels were not significantly different in the two groups (11.3 +/- 1.6 vs. 13.1 +/- 1.2 nmol/L). This suggested that MENT Ac was rapidly converted to MENT in circulation. The hydrolysis of MENT Ac to MENT was confirmed by the in vitro incubation of MENT Ac with blood or plasma and the demonstration of MENT in products following separation by high-performance liquid chromatography (HPLC). Following the demonstration of the safety of MENT Ac in subchronic toxicity studies in rats and rabbits, a pharmacokinetic study was performed in men. In normal men, a single intravenous bolus of 500 micrograms of MENT led to peak serum MENT levels at 3 minutes after dosing (when the first samples were collected), followed by an exponential decline, reaching undetectable levels by 180 minutes. The average terminal half-life and the metabolic clearance rate (MCR) were calculated to be 40 minutes and 2,360 L/day, respectively. The results of the pharmacokinetic studies show that in both men and monkeys, the MCR of MENT is much faster than the values reported for testosterone. The faster MCR can be attributed, in part, to the finding that, in contrast to testosterone, MENT showed no binding to sex hormone binding globulin (SHBG).

7alpha-Methyl-19-nortestosterone (MENT) is a potent synthetic androgen that is resistant to 5alpha-reductases and therefore less prone to over-stimulate the prostate. It is a good candidate for implant administration in long-term androgen replacement therapy for hypogonadal men or as part of a male contraceptive system. To investigate the pharmacokinetics of MENT after i.m. administration, single i.m. injections of 2, 4 or 8 mg of micronized MENT were given in aqueous suspension to 18 healthy men in two clinics. Blood was sampled frequently for 8 h and 1, 2, 3, 4 and 9 days after the injections. Serum MENT concentrations were determined by radioimmunoassay. Peak MENT concentrations were dose-dependent and were reached about 1-2 h after the injections. Doubling the dose of MENT resulted in an increase of 60% in peak serum MENT concentrations. The mean +/- SE clearance rate was 1790 +/- 140 l/day. The antigonadotrophic activity of MENT was investigated by giving six consecutive daily i.m. injections of 1, 2 or 4 mg of MENT to 24 healthy men in two clinics. Blood was sampled before each injection and up to 24 days after the last injection. Serum testosterone and gonadotrophin concentrations (determined by radioimmunoassay and fluoroimmunoassay respectively) decreased in a dose-dependent and statistically significant manner. The highest dose caused a 74% fall in testosterone, a 70% fall in luteinizing hormone, and a 57% fall in follicle stimulating hormone concentrations. MENT injections did not cause any side-effects. The results show that MENT is a potent antigonadotrophic agent in men.

The distribution of testin in the female reproductive system of rats throughout the estrous cycle was examined immunohistochemically. In ovarian follicles, immunostainable testin was localized at the junctions between adjacent granulosa cells. During follicular development, immunostainable testin surrounding the granulosa cells increased in every follicle but was reduced drastically when the follicle was undergoing atresia. Testin was also found in the junctions between adjacent germinal epithelial cells that covered the surface of the ovary, at the lower or the lower lateral borders of each cell. In the uterus and oviduct, immunostainable testin was detected only in the luminal and glandular epithelium, where it formed a polygonal network encircling the apical border of the epithelial cells. During the estrous cycle, there was no drastic change in the distribution of testin in the epithelial cells of the ovary. In the vaginal mucosa, testin was found to be localized only at the junction of the epithelial cells on the surface layer of the stratified epithelium; at different stages of the estrous cycle, distinctive staining for testin could be found at proestrus, metestrus, and diestrus, but not at estrus. It is postulated that testin is a cell junction-associated protein in the female reproductive system.

Leydig and Sertoli cells are both targets of androgen action in the testis. Androgen exerts contrasting effects on the two cell types partially inhibiting steroidogenesis in adult Leydig cell and stimulating adult Sertoli cell functions required to support spermatogenesis. The developmental changes in the messenger RNA (mRNA) levels of androgen receptor (AR) also differ between Leydig and Sertoli cells, with Leydig cell AR mRNA being highest on day 35 postpartum, whereas Sertoli cell AR mRNA levels are highest on day 90. The purpose of the present study was to determine if the concentrations of AR in Leydig and Sertoli cells are differentially regulated during development using quantitative immunostaining. AR protein levels were measured in rat testes after hormonal treatments at three developmental stages: on days 21, 35, and 90 postpartum. At each age, five groups of animals were treated for 4 days with: 1) vehicle; 2) LHRH antagonist (NalGlu, 0.3 mg/kg BW.day) to suppress endogenous levels of androgen that accompany inhibition of LH and FSH secretion; 3) NalGlu + LH (0.2 mg/kg BW.day); 4) NalGlu + testosterone (T, at 7.5 mg/kg BW.day); and 5) NalGlu + MENT (a potent synthetic androgen, 7 alpha-methyl-19-nortestosterone, 0.7 mg/kg BW.day). AR protein was visualized by immunohistochemistry and measured by computer-assisted image analysis in Leydig and Sertoli cells using frozen sections of tests. After NalGlu treatment, AR levels in Leydig cells declined sharply to 42% and 31% of vehicle control (P0.01) in the 21 and 35 days postpartum age groups, respectively, but in 90-day-old rats there was no change. AR levels were partially maintained by exogenous LH, and completely maintained by exogenous androgen treatments in Leydig cells from 21- and 35-day-old rats, whereas in Leydig cells from 90-day-old rats, AR levels were unaffected in all treatment groups. In contrast, after NalGlu treatment, the AR concentration in Sertoli cells from 90-day-old rats were reduced to 32% of control (P0.01). Moreover, in Sertoli cells from 90-day-old rats, AR levels were partially maintained by LH and completely maintained by androgens. A similar trend was observed on day 35. On day 21, however, AR levels in immature Sertoli cells were unaffected in all treatment groups. These results indicate that androgen maximally stimulates AR levels in immature Leydig cells but is without significant effect in adult Leydig cells. In contrast, AR levels in Sertoli cells are more sensitive to androgen regulation in adult compared with immature animals. These findings indicate that there are distinct mechanisms controlling AR concentrations in Leydig and Sertoli cells during the development of the testis.

Anordiol, an antiestrogen, was administered at doses of 2.5 and 10 mg/kg day-1 for 30 and 60 days to adult male rats. A significant decrease in serum LH, FSH, and testosterone levels occurred in the treated animals as compared to controls. Serum testosterone and LH levels decreased to about 15-35% of control value on day 15 of treatment and reached undetected levels thereafter. Serum FSH level also decreased to about 25-50% of control value by day 15 of treatment and remained at this level. Significant decrease in the weights of the tests and accessory reproductive organs occurred following anordiol treatment, while the body weights remained at pretreatment level. Histological examination of the tests revealed significant decrease in the diameter of the seminiferous tubules and in the number of germ cell elements. Spermatogenesis was arrested at the secondary spermatocyte stage. Leydig cells appeared atrophic and contained pyknotic nuclei. The epididymis, prostate, and seminal vesicle showed degenerative changes. The secretory activity of the glandular epithelium of the prostate gland appeared to be markedly reduced. In conclusion, anordiol acts by blocking gonadotropin production and/or release by the pituitary; thereby testosterone production by Leydig cells is not stimulated, causing spermatogenesis arrest.

In 1996, an application was submitted to the US Food and Drug Administration for the use of mifepristone (RU-486) plus the prostaglandin misoprostol in medical abortion. Over 100,000 women in more than 20 countries have received this regimen, which results in pregnancy termination in 92.7-99.0% of treated women. This article presents state-of-the-art information on medical abortion. Reviewed are its pharmacokinetics and metabolism, mechanism of action, and history of use. The article outlines a standard protocol that includes RU-486 administration at the first visit (day 1), misoprostol administration at the second visit (day 3), and post-treatment examination at the third visit (days 14-20) and suggests counseling guidelines. It discusses the contraindications and potential complications of abortifacient agents. Finally, the article compares the experience in the US and Europe of medical versus surgical abortion in terms of effectiveness, complications, and acceptability.

The levonorgestrel-releasing intrauterine system (LNg IUS) is a hormonal contraceptive that is used in the uterine cavity. To determine whether the reasons for choosing LNg IUS vs. copperreleasing intrauterine devices (Cu IUDs) differ and whether their performances are comparable, we carried out a retrospective study in Finland during the first years of LNg IUS use.Gynecological and contraceptive histories of 626 LNg IUS and 626 Cu IUD users and the performance of the device were reviewed from patient records.Women who accepted the LNg IUS were more likely than Cu IUD acceptors to have a history of menstrual bleeding of 6 days or more (44.4% vs. 28.4%), heavy bleeding (44.8% vs. 8.4%) and moderate or severe dysmenorrhea (15.9% vs. 7.5%). In both groups, 70% of the women had used Cu IUDs earlier. However, the LNg IUS acceptors had had more side effects during previous use of CU IUDs (58.2% vs. 28.8%). They also reported more side effects that resulted in discontinuation of a previous Cu IUD (39.4% vs. 10.1%). However, the 12-month life-table continuation rates of 80.6 (SE 1.9)% vs. 83.4 (SE 1.8)% were alike. Cu IUD users discontinued the current method more often because of problems of bleeding and unwanted pregnancy. Among those women who had previously discontinued a hormonal method because of hormonal side effects, there were no differences in the continuation rates of the two groups.LNg IUS can be successfully used by women who cannot use a CU IUD or who have experienced hormonal side effects with oral contraceptives.

The effectiveness of mifepristone, onapristone, and ORG 31806 alone or in combination with anordiol to terminate pregnancy in the rat was evaluated. ORG 31806 at a dose of 2 mg/kg/day, mifepristone at 4 mg/kg/day, and onapristone at 8 mg/kg/day, terminated pregnancy in all treated animals. Anordiol, an antiestrogen, at a dose of 5 mg/kg/day, terminated pregnancy in all treated animals. Anordiol acted synergistically with all three antiprogestins terminating pregnancy in the rat. The antiprogestins at doses that were either partially effective or non-effective became 100% effective when administered with a non-effective dose of anordiol. Thus, combination of ORG 31806 (1 mg/kg/day) plus anordiol (0.31 mg/kg/ day), mifepristone (1 mg/kg/day) plus anordiol (0.62 mg/ kg/day), and onapristone (2 mg/kg/day) plus anordiol (2.5 mg/kg/day) terminated pregnancy in all treated animals. These combinations of the antiprogestins and anordiol decreased significantly the serum progesterone levels but not serum 17 beta-estradiol levels. The present results indicate that the most potent combination was ORG 31806 plus anordiol.The pregnancy termination potency of varying doses of mifepristone, onapristone, and ORG 3806--alone and in combination with the estrogenic/antiestrogenic compound anordiol--was evaluated in adult rats. The antiprogestins and anordiol alone were administered to pregnant female rats on days 7, 8, and 9 of pregnancy and the presence or absence of embryos in utero was determined on day 16. ORG 31806 at a dose of 2 mg/kg/day, mifepristone at 4 mg/kg/day, and onapristone at 8 mg/kg/day terminated pregnancy in 100% of animals; 5 mg/kg/day of anordiol was required. Anordiol acted synergistically with all three antiprogestins. Antiprogestin doses that were either partially effective or ineffective became 100% effective when administered with a noneffective dose of anordiol. The combination of ORG 31806 (1 mg/kg/day) and anordiol (0.31 mg/kg/day) had the most potent pregnancy termination activity. The administration of antiprogestins in combination with anordiol at doses that effectively terminate pregnancy was associated with a significant, persistent reduction in serum progesterone, but no change in serum estradiol levels. The effectiveness of ORG 31806 and anordiol in terminating pregnancy should be evaluated in a non-human primate model to determine its potential clinical use.

To develop a better postcoital contraceptive, the following antiestrogens were tested for their anti-implantation activity in the rat: anordrin, anordiol, tamoxifen, ICI 182,780, and RU 39411. The compounds were administered orally or subcutaneously (s.c.) to female rats on days 1, 2, and 3 of pregnancy. All the antiestrogens tested were 100% effective in preventing blastocyst implantation. The lowest effective doses when administered orally were 10, 1.25, 0.062, 6.0 (partially effective), and 0.01 mg/kg/day, respectively. The estimated median effective doses (ED50) were 5.60, 0.40, 0.035, 5.40, and 0.0074 mg/kg/day, respectively. When administered s.c., the minimum effective doses in preventing blastocyst implantation in all animals were 2.0, 0.1, 0.1, 0.1, and 0.01 mg/kg/day, respectively. Anordrin, anordiol, and ICI 182,780 were more potent when administered s.c.; whereas tamoxifen and RU 39411 were effective at similar doses when administered parenterally or orally. RU 39411 was the most potent among the antiestrogens tested and should be evaluated as a potential postcoital contraceptive. The administration of mifepristone, an antiprogestin, at a dose of 8 mg/kg/day blocked blastocyst implantation in all treated animals; whereas at a dose of 4 mg/kg/day or lower, the drug was ineffective. These findings confirm that estradiol and progesterone are essential for blastocyst implantation in the rat. The capacity of mifepristone to potentiate the anti-implantation activity of the antiestrogens was also determined. The combination of a non-effective dose of each of the antiestrogens (anordrin, anordiol, and tamoxifen), and RU 39411, with mifepristone at a non-effective dose, prevented pregnancy, demonstration that an antiprogestin and antiestrogen act synergistically in blocking blastocyst implantation in the rat. The antiestrogen compounds whose anti-implantation activities were potentiated by mifepristone were found to possess significant estrogenic activity, when assayed by measuring the increase in the uterine weights of ovariectomized rats. The only exception was ICI 182,780, which showed no estrogenic activity in the uterine weight bioassay and did not act synergistically with mifepristone in blocking blastocyst implantation. Estradiol was effective in preventing pregnancy at a dose of 1 microgram/kg/day. The combination of non-effective doses of estradiol and mifepristone did not prevent pregnancy. The findings that mifepristone potentiates the anti-implantation activity suggests that the synergistic effect may be a unique property of this class of antiestrogens.In New York, female and male rats copulated on the afternoon of proestrus as part of a study aiming to determine whether antiestrogens alone or in combination with mifepristone (RU-486) will block blastocyst implantation in the rat. This study is part of research efforts to develop a better postcoital contraceptive. The antiestrogens included RU39411; ICI 182,780; anordrin; anordiol; tamoxifen; and estradiol. The laboratory researchers treated the rats orally or subcutaneously on days 1, 2, and 3 of pregnancy. They killed them on day 8-9 to examine the uteri for the presence or absence of implanted embryos. All the antiestrogens effectively prevented blastocyte implantation. Using the measurement mg/kg/day, the lowest effective oral dose was 10 for anordrin; 1.25 for anordiol; 0.062 for tamoxifen; 6 (80% effective) for ICI 182,780; and 0.01 for RU 39411. These antiestrogens' estimated median effective doses were 5.6, 0.4, 0.035, 5.4, and 0.0074 mg/kg/day, respectively. In all animals, the minimum effective doses administered subcutaneously were 2, 0.1, 0.1, 0.1, and 0.01 mg/kg/day, respectively. Anordrin, anordiol, and ICI 182,780 were more effective during subcutaneous administration while tamoxifen and RU 39411 were as effective at similar doses during parenteral or oral administration. The most potent antiestrogen was RU 39411. This antiestrogen should be evaluated as a potential postcoital contraceptive. An 8 mg/kg/day dose of RU-486 blocked blastocyst implantation in all rats. The 4 mg/kg/day dose was completely ineffective. RU-486 augmented the activity of anordrin, anordiol, tamoxifen, and RU39411 synergistically, resulting in prevention of pregnancy at non-effective doses of RU-486 and the antiestrogens. These same antiestrogens also exhibited significant estrogenic activity as determined by an increase in uterine weights of ovariectomized rats. Estradiol prevented pregnancy at a dose of 1 mcg/kg/day. RU-486 did not potentiate estradiol. These findings suggest that the synergistic effect of anordrin, anordiol, tamoxifen, and RU39411 may be unique to these antiestrogens.

A biological factor that inhibits the in vitro secretion of testin by Sertoli cells was purified to apparent homogeneity from conditioned medium of germ cells isolated using trypsin. Partial N-terminal amino acid sequence analysis of the purified germ cell factor revealed a sequence of NH2-IVGGYTXAAN. Comparison of the sequence with the existing protein database revealed that it is homologous to trypsin. Immunoprecipitation experiments using either [35S]-labeled germ or Sertoli cell proteins and a monospecific anti-trypsin antibody failed to demonstrate the synthesis and secretion of trypsin by these testicular cells, suggesting the isolated factor is the residuary trypsin that was used for isolating germ cells from seminiferous tubules. Subsequent experiments revealed that trypsin per se can inhibit the secretion of Sertoli cell testin and clusterin dose-dependently, whose effect can be prohibited by soybean trypsin inhibitor (STI). In view of these findings, a nonenzymatic procedure was deemed necessary to prepare germ cell conditioned medium (GCCM) to assess whether an authentic biological factor(s) is indeed present. Four batches of conditioned medium of germ cells isolated by a mechanical procedure without the use of trypsin were fractionated by sequential Mono Q anion exchange and C8 reversed-phase HPLC. When these fractions were monitored for testin modulatory activity using an in vitro bioassay with primary cultures of Sertoli cells, it was shown that GCCM prepared by this procedure indeed contained testin modulatory bioactivity. Since testin is a novel component of specialized junctions between Sertoli and germ cells, the identification of a germ cell factor(s) that affects its secretion by Sertoli cells suggests a dynamic biochemical relationship between these cell types in the seminiferous epithelium.

To investigate the mechanism of androgen-induced thymolysis, the effects of various androgens, including testosterone (T), 19-nortestosterone, and 7 alpha-methyl-19-nortestosterone (MENT), were compared with those of estradiol and dexamethasone (DEX) in intact, castrated, and adrenalectomized male rats. The potency comparisons on thymus regression, based on mass of steroids, showed DEX to be the most potent, followed by estradiol and the androgens. Among the androgens, MENT was the most potent, followed by nortestosterone and T, an order similar to their anabolic potency on muscle. As the thymolytic effects of T and MENT were not altered by the concomitant administration of an aromatase inhibitor or a 5-reductase inhibitor, it was concluded that the effects of androgens were not mediated by their conversion to estrogens or 5 alpha-reduced metabolites. Involvement of glucocorticoid receptors in androgen action was excluded because mifepristone (an antiglucocorticoid) blocked DEX-induced, but not T- or MENT-induced, thymus regression. Flutamide, an antiandrogen, significantly blocked the thymolytic effect of T and MENT, providing further support for this conclusion. This suggested that the thymolytic action of androgens is an intrinsic property mediated via androgen receptors (AR). The occurrence of AR in the thymus was demonstrated by binding assays and the presence of AR messenger RNA (mRNA) by reverse transcriptase-polymerase chain reaction. Quantitative reverse transcriptase-polymerase chain reaction for AR mRNA in the thymus showed 6-fold more AR mRNA in the thymic epithelial cells than in the thymocytes. However, epithelial cells represent only a small fraction of the thymus. Hence, it is hypothesized that the androgens produce their thymolytic effects by stimulating the secretion of a factor(s) by the thymic epithelial cells that, in turn, causes regression of the thymus.

Testosterone produced by Leydig cells is critical for the maintenance of spermatogenesis by Sertoli cells throughout adulthood in the rat. However, the presence of androgen receptors (AR) in Leydig cells in prepubertal rats suggests additional roles for androgen in early Leydig cell function and differentiation. In the present study, AR messenger RNA (mRNA) was directly measured by in situ hybridization in sections of rat testes at three developmental stages: on day 21 postpartum, when Leydig cells exist as mesenchymal-like progenitors; on day 35, when they are still immature, producing low amounts of testosterone; and on day 90, when they are fully functional in the sexually mature animal. Testicular AR mRNA was detected in Leydig cells, pericytes, peritubular myoid cells, and Sertoli cells. On day 90, AR mRNA levels in Sertoli cells varied with the cycle of the seminiferous epithelium, achieving peak intensity at stages VII-VIII. Measurements were made by image analysis and expressed as integrated signal intensities per unit labeled area (mean +/- SEM; n = 3 rats at each age). The results showed that levels of Leydig cell and Sertoli cell AR mRNA change significantly during development (P < 0.05). Leydig cell AR mRNA was intermediate on day 21 (at 17.3 +/- 0.7), highest on day 35 (at 26.9 +/- 1.6), and lowest on day 90 (at 11.8 +/- 1.1). The trend for isolated Leydig cells from these three ages was identical. In contrast, Sertoli cell AR mRNA was lowest on day 21 (at 19.3 +/- 1.0), intermediate on day 35 (at 24.5 +/- 1.4), and highest on day 90 (at 36.9 +/- 0.5). In Leydig cells, the highest level of AR mRNA was present during puberty, whereas the greatest amount of AR mRNA in Sertoli cells was present on day 90. This indicates that Leydig cells and Sertoli cells use different mechanisms to maintain AR levels. We infer from these data that Leydig cells are maximally sensitive to androgen during puberty, which is consistent with our hypothesis that androgens facilitate their differentiation.

Testosterone, the principal androgen secreted by Leydig cells, exerts a wide range of actions including growth of the male reproductive tract (androgenic effects) and growth of non-reproductive tissues such as muscle, kidney, liver, and salivary gland (anabolic effects). As androgenic steroids were discovered some were found to have relatively more anabolic than androgenic activity. The results reviewed in this report suggest that these differences result, in part, from the differential metabolism of the steroids in individual tissues and the varied activities of the individual metabolites. In the accessory sex organs (e.g. the prostate) testosterone is 5 alpha-reduced to dihydrotestosterone (DHT) which, due to its higher affinity for androgen receptors (AR), amplifies the action of testosterone. In contrast, when 19-nortestosterone (NT) is 5 alpha-reduced, its affinity for AR decreases, resulting in a decrease in its androgenic potency. However, their anabolic potency remains unchanged since significant 5 alpha-reduction of the steroids does not occur in the muscle. 7 alpha-methyl-19-nortestosterone (MENT) does not get 5 alpha-reduced due to steric hindrance from the 7 alpha-methyl group. Therefore, the androgenic potency of MENT is not amplified as happens with testosterone. These metabolic differences are responsible for the increased anabolic activity of NT and MENT compared to testosterone. Part of the biological effects of testosterone are mediated by its aromatization to estrogens. The fact that MENT is also aromatized to 7 alpha-methyl estradiol, a potent estrogen, in vitro by human placental and rat ovarian aromatase suggests that some of the anabolic actions of MENT may be mediated by this estrogen.

Testin I and testin II are the two molecular variants of testin that are synthesized and secreted by Sertoli cells in vitro. N-Terminal and partial internal amino acid sequence analysis of testin I and testin II reveals that these molecules are identical with the exception that testin II has three extra N-terminal amino acids of TAP compared to testin I. Studies using immunohistochemistry suggested that testin is a component of the specialized junctional complexes in the seminiferous epithelium and other tissues. Immunoreactive testin is localized not only at Sertoli-Sertoli and Sertoli-germ cell junctions, but also at sites of similar junctions in the liver, epididymis, kidney, and intestine. Other physiological studies have shown that the secretion of testin is tightly coupled to the presence of germ cells. In view of its possible role in germ cell development and its unique localization in the cell junction, the purpose of the present study was to determine the structure of testin by sequencing its full-length cDNA. Two synthetic degenerate oligonucleotides based on the N-terminal and an internal amino acid sequence were used for polymerase chain reaction (PCR) to obtain a 289-bp cDNA fragment. This PCR product was subsequently used to isolate a 1371-bp cDNA from a cDNA expression library constructed from Sertoli cell poly(A) RNA. This cDNA coded for a 333 amino acid peptide that starts with an ATG initiation codon from the 5' end and ends with a TGA termination codon located 245 nucleotides before the polyadenylation site. The deduced amino acid sequence indicates that testin contains a 16 amino acid signal peptide with two possible cleavage sites that yield 314 and 317 amino acids for testin I and testin II with calculated molecular weights of 36,029 and 36,299, respectively. Comparison of the entire coding region of testin with existing sequences at Genbank, EMBL, and Protein Identification Resource indicates that testin shares 58%, 57.4%, and 61% identity with rat, mouse, and human cathepsin L at the amino acid level, respectively. The positions of all of the 7 Cys residues and 8 of the 10 Trp residues in testin are conserved with respect to those present in cathepsin L. It is noted that Cys-122 in the predicted active site of cathepsin L was replaced with Ser-122 in testin. In view of the striking primary sequence homology between testin and cathepsin L, we assayed the proteolytic activity of testin using conditions known to activate cathepsin L.(ABSTRACT TRUNCATED AT 400 WORDS)