Use of the granulosa cell aromatase bioassay for measurement of bioactive follicle-stimulating hormone in urine and serum samples of diverse species

Ovarian steroids and growth factors are intragonadal modulators which augment a key endpoint of follicle-stimulating hormone (FSH) action in granulosa cells: the induction of aromatase activity. Studies of these paracrine hormones that enhance FSH-stimulated estrogen biosynthesis by cultured rat granulosa cells, have led to the development of a sensitive and specific bioassay for FSH. This newly developed granulosa cell aromatase bioassay (GAB) allows for the measurement of bioactive FSH levels in serum and urine of humans and animals with various physiological and pathological conditions. These studies have demonstrated that the GAB assay is useful in detecting possible changes in the molecular forms of FSH. The adaptation of this method for urine samples allows for the measurement of bio-FSH levels in situations where venipuncture is not practical or in species for which specific radioimmunoassays are not available.     

Immunohistochemical localization of tissue-type plasminogen activator in ovaries before and after induced and spontaneous ovulation in the rat

Summary The observation that tissue-type plasminogen activator (tPA) activity increased dramatically in preovulatory follicles has led to the hypothesis that plasminogen activation is causally related to follicle rupture. With immunohistochemistry, we have studied the appearance of tPA in ovaries of immature rats induced to ovulate and in adult cycling rats. Treatment of immature female rats with a single dose of pregnant mare serum gonadotropin (PMSG) induced follicular maturation. A subsequent human chorionic gonadotropin (hCG) injection resulted in follicle rupture 12–14 h later. PMSG treatment alone did not induce appearance of tPA-immunoreactive cells in any ovarian compartment. After hCG stimulation, however, theca cells, granulosa cells, and oocytes of pre- and postovulatory follicles displayed distinct tPA immunoreactivity. Fibroblastlike cells in the theca layers and tunica albuginea of the follicle apex also demonstrated localized cytoplasmic tPA reactivity. In addition to tPA synthesis in preovulatory follicles, hCG also induced tPA staining in the theca (but not granulosa) layers of non-ovulatory follicles. At 24 h after hCG treatment, there was a marked tPA staining in developing corpora lutea, ovulated ova, and oviductal epithelium. Ovaries from regularly cycling adult rats displayed a similar ovulation-related pattern of tPA immunostaining. The appearance of tPA in different cell types of the preovulatory follicle and in the fibroblast-like cells at the follicle apex, strengthens the hypothesis of a direct involvement of tPA in follicle rupture. Presence of tPA in postovulatory oocytes, cumulus cells, and surrounding oviductal epithelium may also indicate a role for tPA in the transfer of eggs in the oviduct.This work was supported by NIH Research Grants HD-14084; 12303     

Effect of transforming growth factor-beta on human chorionic gonadotropin induced testosterone production by cultured rat testicular cells

The potential role of transforming growth factor-beta (TGF-beta) as an intragonadal regulator in the testis was investigated by studying the effect of TGF-beta on testosterone (T) production by neonatal rat testis cells in primary cultures. After 3 days of preincubation in serum-free medium, testis cells were treated with hormones for 3 additional days. Human chorionic gonadotropin (hCG) treatment (0.3-30 ng/ml) of testis cells elicited a dose-dependent increase of T levels with maximum values greater than 9-fold over baseline. Although TGF-beta alone did not affect T levels, a dose-dependent inhibition of hCG-stimulated T production was observed when cells were cotreated with TGF-beta. Maximal inhibition was greater than 85%, and the IC50 value was 5 ng/ml (2 x 10(-10) M; n = 5 experiments). This inhibitory effect was evident 48 h after the initiation of treatment and could be reversed 1 day after the cessation of TGF-beta exposure of cells. TGF-beta also reduced forskolin and (Bu)2cAMP-induced T production (greater than 85% decrease), indicating that TGF-beta can inhibit steroidogenesis distal to the formation of cAMP. The conversion of exogenously added androgen precursors (progesterone (P) and 17 alpha-hydroxyprogesterone) to T by hCG-stimulated cells was suppressed by the addition of TGF-beta. In contrast, endogenous P accumulation did not change in cultures treated with TGF-beta. Because TGF-beta-like activity has been found in the testis, the observed inhibitory effect of TGF-beta suggests a potential intratesticular regulatory role of this growth factor.     

Rapid changes in the expression of inhibin alpha-, beta A-, and beta B-subunits in ovarian cell types during the rat estrous cycle

Distributions of inhibin alpha-, beta A-, and beta B-subunits in different ovarian compartments were studied in cycling female rats by in situ hybridization with complementary RNA probes and using immunohistochemical localization with antibodies selective for each inhibin subunit. Consistent with earlier studies showing inhibin production by granulosa cells of maturing follicles, we also detected mRNAs for inhibin alpha-, beta A-, and beta B-subunits in granulosa cells of these follicles. However, based on immunohistochemistry and in situ hybridization, we found that inhibin alpha- is not only expressed in granulosa cells of mature follicles but in follicles at all stages of maturation, including primary to tertiary follicles. A number of primordial follicles also contained alpha mRNA and immunodetectable alpha-subunit. Interestingly, theca interna and interstitial gland cells contained inhibin alpha mRNA and alpha-subunit. Low levels of inhibin alpha immunoreactivity as well as specific hybridization to the complementary inhibin alpha mRNA probe were observed in newly formed luteal tissue. beta-Subunits, on the other hand, were detected exclusively in granulosa cells of healthy tertiary follicles. The changes in expression of inhibin alpha-, beta A-, and beta B-subunits were more pronounced during the follicular phase of the cycle: inhibin alpha reached its highest level in granulosa cells, theca interna, and interstitial gland cells a few hours after the LH/FSH surge, while at the same time the beta-subunits decreased dramatically in granulosa cells of mature follicles. Immediately before ovulation (estrus 0200 h), the alpha-subunit sharply declined in preovulatory follicles and was present mainly in granulosa cells from nonovulatory follicles at various stages of maturation. At that time, the beta A- and beta B-subunits could not be detected in preovulatory follicles but were localized mainly in small tertiary follicles (less than 300 microns). Unlike for the alpha- and beta B-subunits, beta A mRNA and immunoreactivity was present in large tertiary follicles (approximately 600 microns) immediately before ovulation. The present findings support the hypothesis that a decrease in inhibin production could be responsible for the secondary FSH surge observed early on estrus. This could be initiated by a change in the ratios of activin-inhibin production by decreasing first, the levels of beta-subunits, second, the levels of alpha-subunit, and third, by a resurgence of activin A produced mainly by granulosa cells from large tertiary follicles.(ABSTRACT TRUNCATED AT 400 WORDS)     

Epidermal growth factor and basic fibroblast growth factor suppress the spontaneous onset of apoptosis in cultured rat ovarian granulosa cells and follicles by a tyrosine kinase-dependent mechanism.

Recent biochemical studies have suggested that apoptotic cell death is the molecular mechanism underlying the degeneration of ovarian follicles during atresia. Using a sensitive autoradiographic method for the detection of DNA fragmentation, we studied apoptosis in ovarian granulosa cells or intact follicles placed in serum-free culture as model systems to elucidate the hormonal regulation of atresia. Immature rats (25 days old) were primed for 2 days with 10 IU equine CG to induce a homogeneous population of mature preovulatory follicles. Granulosa cells isolated from these follicles contained predominantly intact high mol wt DNA. However, a time-dependent, spontaneous onset of internucleosomal DNA fragmentation characteristic of apoptotic cell death occurred in granulosa cells during culture. Treatment of granulosa cells with epidermal growth factor (EGF), transforming growth factor-alpha (TGF alpha), or basic fibroblast growth factor (bFGF) inhibited the spontaneous onset of apoptotic DNA cleavage found during culture by 40-60%. In contrast, insulin-like growth factor I, insulin, TGF beta and tumor necrosis factor-alpha were ineffective. Likewise, activation of the protein kinase A or C pathways with forskolin or phorbol 12-myristate 13-acetate, respectively, did not prevent the onset of DNA fragmentation, although inclusion of a tyrosine kinase inhibitor (genistein) completely blocked the ability of EGF, TGF alpha, and bFGF to suppress apoptosis in granulosa cells. Similar to cultured granulosa cells, a spontaneous onset of apoptosis was also observed to occur in isolated preovulatory follicles during culture. Furthermore, treatment of follicles with EGF or bFGF inhibited the spontaneous initiation of apoptosis, and the suppressive effects of these growth factors were also attenuated by co-treatment with genistein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of human immunodeficiency virus replication in acutely infected CD4+ cells by CD8+ cells involves a noncytotoxic mechanism.

The mechanism by which CD8+ T cells from human immunodeficiency virus (HIV)-infected individuals suppress HIV replication in acutely infected CD4+ T cells was investigated. Cytotoxicity was not involved, as the antiviral activity of the CD8+ cells did not correlate with the ability to lyse HIV-infected or uninfected CD4+ T cells. In addition, the frequency of HIV-infected CD4+ cells increased during coculture with CD8+ T cells even in the absence of detectable levels of virus replication. Moreover, separation of the CD4+ and CD8+ cells by a 0.4-micron-pore-size filter delayed HIV replication, indicating a role, at least in part, for a soluble factor. However, cell contact was required for optimal antiviral activity. These results extend further the observation on the mechanism of antiviral HIV activity by CD8+ cells from infected individuals. They support the conclusion that CD8+ cells can play a major role in preventing development of disease in HIV-infected individuals.     

The rat liver asialoglycoprotein receptor polypeptide must be inserted into a microsome to achieve its active conformation

Affinity chromatography on galactose-Sepharose has been utilized to demonstrate that rat liver asialoglycoprotein receptor synthesized in vitro in a reticulocyte lysate system is capable of binding carbohydrate ligand only when dog pancreas microsomes are present during translation. Analysis of receptor isolated from tunicamycin-treated rat hepatocytes indicates that glycosylation is not necessary for receptor activity. Genetically engineered receptor derivatives in which the natural membrane anchor is either deleted entirely or replaced with a cleavable signal sequence derived from dog preproinsulin have been used to demonstrate that: (a) inactive receptor made in the absence of membranes does not result from incorrect nucleation of folding around the hydrophobic portion of the polypeptide which is normally buried in the membrane and (b) the carbohydrate-binding domain of the receptor does not need to be tethered to the luminal side of the membrane to fold correctly. These results suggest that factors within the lumen of the microsomes are essential to establish the native conformation of the binding domain.     

Autocrine role of estrogens in the augmentation of luteinizing hormone receptor formation in cultured rat granulosa cells

The effects of estrogens on gonadotropin-stimulated luteinizing hormone (LH) receptor formation were examined in primary cultures of rat granulosa cells. Granulosa cells were cultured for 3 days with increasing concentrations of follicle-stimulating hormone (FSH) in the presence or absence of native and synthetic estrogens. Follicle-stimulating hormone stimulated LH receptor formation in a dose-dependent fashion, and estrogens enhanced the FSH-stimulated LH receptor content by decreasing the apparent ED50 of FSH. At 6.25 ng/ml FSH, the enhancement in LH receptor was estrogen dose dependent, with an ED50 value of about 3 X 10(-9) M for 17 beta-estradiol. The increased LH receptor content seen in cells treated with FSH and estrogen was correlated with increased cAMP production by these cells in response to LH stimulation. Time course studies revealed enhancement of FSH-stimulated LH receptor induction at 48 and 72 h of culture. Granulosa cells were also cultured with FSH for 2 days to induce functional LH receptors, then further cultured for 3 days with LH in the presence or absence of estrogens. At 30 ng/ml LH, increasing concentrations of estrogens maintained LH receptor content in a dose-dependent fashion, with their relative estrogenic potencies in keeping with reported binding affinities to estrogen receptors. An autocrine role of estrogens on LH receptor formation was further tested in granulosa cells treated with FSH and an aromatase substrate (androstenedione) to increase estrogen biosynthesis. Cotreatment with semipurified estrogen antibodies partially blocked the FSH stimulation of LH receptors, whereas nonimmune serum was ineffective. Also, inclusion of diethylstilbestrol prevented the inhibitory effect of the estrogen antibodies. Thus, local estrogens in ovarian follicles may play an autocrine role in granulosa cells to enhance LH receptor formation and to increase granulosa cell responsiveness to the LH surge, with subsequent ovulation and adequate corpus luteum formation.     

Regulation of 3 beta-hydroxysteroid dehydrogenase activity by human chorionic gonadotropin, androgens, and anti-androgens in cultured testicular cells

delta 5-3 beta-Hydroxysteroid dehydrogenase is a key enzyme for testicular androgen biosynthesis and a marker for the Leydig cells. The hormonal regulation of this enzyme was studied in cultured rat testicular cells. Human chorionic gonadotropin (hCG) increased testosterone production in vitro while time course studies indicated a biphasic action of the gonadotropin on 3 beta-hydroxysteroid dehydrogenase activity. An initial stimulation (51%) of the enzyme was detected between 3 and 12 h of culture when medium testosterone was low. This is followed by an inhibition of 3 beta-hydroxysteroid dehydrogenase activity on days 2 and 3 of culture when medium testosterone was elevated. Concomitant treatment with a synthetic androgen (R1881) inhibited 3 beta-hydroxysteroid dehydrogenase activity and testosterone production in hCG-treated cultures while an anti-androgen (cyproterone acetate) increased 3 beta-hydroxysteroid dehydrogenase activity and testosterone biosynthesis. Addition of 10(-5) M spironolactone, an inhibitor of 17 alpha-hydroxylase, blocked the hCG stimulation of testosterone production but increased medium progesterone. In the absence of the secreted androgen, hCG stimulated 3 beta-hydroxysteroid dehydrogenase activity in a time- and dose-related manner. Furthermore, hCG stimulation of 3 beta-hydroxysteroid dehydrogenase activity and progesterone accumulation in spironolactone-supplemented cultures was decreased by concomitant treatment with R1881 but was not affected by cyproterone acetate. The inhibitory effect of R1881 was blocked by the anti-androgen. In the absence of hCG, treatment with testosterone, dihydrotestosterone, or R1881, but not promegestone, alone also inhibited 3 beta-hydroxysteroid dehydrogenase activity while the inhibitory effect of testosterone was blocked by cyproterone acetate. Thus, hCG stimulates 3 beta-hydroxysteroid dehydrogenase activity in cultured testicular cells. The androgenic steroidogenic end products, in turn, inhibit this enzyme. The hormonal regulation of 3 beta-hydroxysteroid dehydrogenase activity may be important in the ultrashort loop autoregulation of androgen biosynthesis.     

Thermal denaturation of calf thymus DNA: existence of a GC-richer fraction

In 2.5 x 10(-4)M EDTA buffer, the derivative melting curve of calf thymus DNA shows a major band at 47 degrees with a shoulder at about 54 degrees . The fraction of melting area of this shoulder is about 13%. For reconstituted polylysine-calf thymus DNA complexes, in addition to the melting of free DNA regions at about 50 degrees (T(m)) there is another melting at about 106 degrees (T(m)) of polylysine-bound regions. The melting band of the complex at T(m) is not symmetrical. As more polylysine is bound to DNA the melting amplitude is diminished greatly on the major band at 47 degrees but only slightly on the shoulder at 54 degrees . The insensitivity of this shoulder appears to result from the existence of a 13% fraction of calf thymus DNA containing 55% GC. It is not favorably bound by polylysine. It remains in the supernatant after centrifugation and melts at about 54-56 degrees . This conclusion is further supported by two facts: the reconstitution method provides a condition for selective binding of polylysine to AT-rich DNA, and it yields a fully symmetric melting band at T(m) for complexes of polylysine with homogeneous bacterial DNA such as the one from M. luteus.