N-cadherin mediates Sertoli cell-spermatogenic cell adhesion.

The complex topological association of Sertoli cells and spermatogenic cells in the testis suggests the existence of cell surface adhesion molecules that regulate cellular interactions within the seminiferous epithelium. The recent report of N-cadherin mRNA expression in the mouse testis implies the involvement of this known adhesion molecule in testicular cell binding. Accordingly, here we report that (1) N-cadherin is found on the surface membranes of rat spermatogenic cells and on Sertoli cells, and (2) that N-cadherin is a partial mediator of Sertoli cell-germ cell adhesion as tested in an vitro cell-cell binding assay. Antiserum directed against the N-cadherin cell adhesion recognition sequence was used for Western blot anlaysis of purified plasma membranes from Sertoli cells and from spermatogenic cells. Both membrane preparations exhibited reactivity at an appropriate M(r) of about 130 kDa. In addition, immunofluorescence assays demonstrated that both germ cells and Sertoli cells were labeled by anti-N-cadherin. Finally, the antiserum was included in a cytometer-assisted cell-cell binding test to determine its inhibitory ability. The antiserum consistently reduced specific testicular cell-cell adhesion by 30%-50%. This is the first demonstration that antibodies directed against the cadherin cell adhesion recognition sequence are capable of inhibiting cell-cell interactions. Pre-incubation of either rat Sertoli cells or spermatogenic cells alone was sufficient to achieve statistically significant inhibition of intercellular adhesion. We conclude, therefore, that N-cadherin is expressed by both Sertoli cells and spermatogenic cells and that N-cadherin is one of a number of regulatory molecules mediating local cellular associations in the mammalian seminiferous tubule.

Germ cells of the mouse testis express P450 aromatase.

Estrogen production within the testis has been a subject of considerable controversy for many years. Several studies have shown that both Sertoli and Leydig cells produce estrogen during different stages of development. Therefore, we have conducted experiments to localize aromatase, a cytochrome P450 enzyme that converts androgen to estrogen, within the testis. First, P450 aromatase (P450arom) was localized in germ cells of the adult mouse testis by immunocytochemistry, using an antiserum generated against purified human placental cytochrome P450arom. In the germinal epithelium, P450arom was located primarily in the Golgi region of round spermatids, throughout the cytoplasm of elongating spermatids, and along the flagella of late spermatids. Second, localization of P450arom within the germinal epithelium was supported by Western blot analysis of isolated germ cells. Third, Northern blot analysis using a mouse P450arom cDNA probe indicated that the mRNA for the mouse P450arom was present in testicular germ cells. Fourth, P450arom activity was measured in germ cells by the 3H2O water assay. Based upon these observations, we conclude that germ cells are a site of estrogen synthesis in the adult mouse testis.

In vitro effects of psychoactive and non-psychoactive cannabinoids on immature rat Sertoli cell function.

We have examined the effects of psychoactive and non-psychoactive cannabinoids on isolated immature rat Sertoli cells cultured in either serum-free or serum-containing media. Lactate accumulation by Sertoli cells in serum-free control cultures was 10 fold greater than the values obtained in control cultures exposed to serum. Under either culture condition, 3.1 micrograms/ml of delta-9-tetrahydrocannabinol (THC) or cannabinol (CBN) stimulated lactate secretion above control levels. Cannabidiol (CBD) stimulated lactate secretion in serum-containing but not in serum-free media. Using serum-free culture conditions, we next studied the in vitro effects of combinations of THC and either epinephrine or FSH on lactate and transferrin secretion by immature rat Sertoli cells. Co-incubation of 0.1 microM epinephrine with 0.8 or 3.1 microgram/ml THC significantly stimulated lactate secretion when compared to epinephrine or THC alone, while only the high THC dose increased transferrin secretion. Moreover, co-incubation of FSH (1 micrograms/ml) with THC (0.8 or 3.1 micrograms/ml), significantly stimulated both lactate and transferrin production by immature rat Sertoli cells. These results add to the growing evidence that cannabinoids can exert direct effects on Sertoli cell function and modulate their responses to physiological stimuli.

Effects of age, photoperiod and follicle-stimulating hormone on lactate production by cultured Sertoli cells from prepubertal Siberian hamsters (Phodopus sungorus).

To define a functional difference in Sertoli cells of animals exposed to different photoperiodic conditions, we isolated Sertoli cells from the testes of juvenile Siberian hamsters and cultured them in serum-free medium. In all age groups studied, Sertoli cells isolated from hamsters with delayed and normal puberty responded to follicle-stimulating hormone (FSH) with an increase in lactate production. The increase in lactate production induced by 1000 ng FSH ml-1 was significantly greater in Sertoli cells isolated from hamsters with delayed puberty than in those with normal puberty. These results suggest that Sertoli cells of Siberian hamsters exposed to short photoperiod in vivo may respond to increases in plasma FSH concentrations associated with photostimulation or spontaneous sexual maturation by an increase in secretory activity that may be critical for the initiation of spermatogenesis.

Sertoli cell plasma membrane polypeptides involved in spermatogenic cell-Sertoli cell adhesion.

This study concerns Sertoli cell-spermatogenic cell adhesive interactions in the seminiferous tubule. Sertoli cell surface polypeptides involved in germ cell-Sertoli cell adhesion were identified by serological inhibition of an in vitro Sertoli-germ cell adhesion assay. This assay was modified from a previously reported adhesion assay, and employs a scanning laser cytometer for quantification of adherent cells. Reactivity of the polyclonal antiserum raised against rat Sertoli cells was also assessed via immunofluorescent microscopy. The addition of antiserum to the adhesion assay resulted in a 42% to 66% inhibition of cell-cell adhesion. Moreover, preincubation of antiserum with Sertoli cell monolayers resulted in a significant reduction of spermatogenic cell binding. Conversely, preincubation of antiserum with germ cells resulted in no reduction. Western blot analysis of the antiserum against purified Sertoli cell membranes indicated reactivity with four polypeptides. The data suggest that one or more of these polypeptides are directly involved in the adhesion of germ cells to Sertoli cell monolayers in vitro.

Adaptive fuzzy leader clustering of complex data sets in pattern recognition.

A modular, unsupervised neural network architecture that can be used for clustering and classification of complex data sets is presented. The adaptive fuzzy leader clustering (AFLC) architecture is a hybrid neural-fuzzy system that learns online in a stable and efficient manner. The system used a control structure similar to that found in the adaptive resonance theory (ART-1) network to identify the cluster centers initially. The initial classification of an input takes place in a two-stage process: a simple competitive stage and a distance metric comparison stage. The cluster prototypes are then incrementally updated by relocating the centroid position from fuzzy C-means (FCM) system equations for the centroids and the membership values. The operational characteristics of AFLC and the critical parameters involved in its operation are discussed. The AFLC algorithm is applied to the Anderson iris data and laser-luminescent finger image data. The AFLC algorithm successfully classifies features extracted from real data, discrete or continuous, indicating the potential strength of this new clustering algorithm in analyzing complex data sets.

Quantification of an in vitro cell-cell adhesion assay using interactive laser scanning cytometry.

We are interested in identifying cell-cell adhesion molecules on the surface of Sertoli cells that mediate Sertoli cell-spermatogenic cell adhesion. Numerous cell-cell adhesion assays employ microscopic observation, photomicroscopy or radioactive isotopes for quantification. Previously, we developed an in vitro assay for testicular cell interactions. This assay was, however, time consuming using photography for analysis. We have now modified this system using laser cytometry to quantify adherent cells. Rat testicular epithelial cells are cultured for approximately 6 days before labelling with fluorescein diactetate (FDA) to assess confluency by image scanning so that spermatogenic cell binding can be normalized to available epithelial cell surface area. Rat spermatogenic cells are labeled with FDA before addition to epithelial cell monolayers. In some studies, purified spermatogenic cell populations were isolated to determine average cell size. We found that spermatocyte area varied between 225-500 microns2, spermatids were 100-225 microns2 and residual bodies were less than 100 microns2. Using these parameters, scanning cytometry allows the differential analysis of adhesion by individual germ cell sub-classes from mixed cell suspensions, saving time, animals, and major expense. The scanning laser assisted assay is faster, more reproducible and less subjective than earlier cell-cell adhesion assays using light microscopy or isotopes. This experimental approach should facilitate any cell-cell adhesion assay in which one cell type is adherent to a substrate.

Evidence for a direct anterior pituitary site of delta-9-tetrahydrocannabinol action.

The effects of delta-9-tetrahydrocannabinol (THC), alone and in the presence of estradiol (E), on several estrogen-sensitive parameters in the immature female rat were examined, and it was demonstrated that THC administration antagonized the stimulatory effects of E on anterior pituitary weight and on both the secretion and pituitary content of prolactin. In the current study, the anterior pituitary gland was examined as a potential site of THC action in the ability of this cannabinoid to antagonize E-induced stimulation of pituitary function. A stimulatory dose of E (1 nM) significantly elevated prolactin levels in pituitary cells derived from either immature or retired breeder animals. Whereas THC (1 microM) alone had no effect on prolactin levels when compared to controls, THC completely prevented the E-induced increase in media prolactin levels. Moreover, THC blocked the ability of E to desensitize pituitary cells to the inhibitory influence of dopamine. Together with the findings that THC inhibited E-induced stimulation of total RNA synthesis in pituitary cell cultures, these data strongly suggest that THC antagonizes the stimulatory effect of E on the pituitary by a direct action at the adenohypophyseal level.

Effects of elevated gonadotropin levels on thyroid function in the male rat.

In order to determine if thyroid disease in hypogonadal men is the result of chronic elevation of serum gonadotropins, thyroid histology and serum thyroid hormone levels were evaluated in male rats that had been castrated either 2 weeks or 15 months previously. Despite significantly elevating serum LH levels, castration did not affect thyroid structure or function. Serum total T4 levels were reduced with age in both short and long-term castrate animals but returned to the levels seen in young rats when testosterone was replaced. Testosterone replacement also increased free T4 levels in both the young and old castrate rats. Neither age nor testosterone replacement affected serum T3 or TSH levels.