Primary culture of purified Leydig cells isolated from adult rat testes

Methods for isolating highly purified Leydig cells permit the study of acute responses and biochemical properties of Leydig cells independent of other testicular cell types. The present study describes the development of a primary culture system for purified Leydig cells from adult rats in which the cells retain their ability to secrete testosterone for at least 72 h in culture. When Leydig cells were cultured in tissue culture medium 199--0.1% BSA (M199-BSA), basal testosterone secretion declined by 72 h, whereas hCGB-stimulated testosterone secretion was reduced by 48 h. Changing the culture medium twice daily or adding 0.5% fetal calf serum (fcs) enhanced basal and gonadotropin-stimulated testosterone secretion at 72 h in culture, although responsiveness to hCG was reduced to 57% of that in freshly isolated cells. Incubation of Leydig cells in the defined culture medium Dulbecco's Modified Eagles-Ham's F-12 (1:1, vol/vol) supplemented with 15 mM Hepes buffer, transferrin, insulin, and epidermal growth factor (DHG:F12 + Hepes + TIE) in either the presence or absence of 0.5% fcs yielded functional Leydig cells for longer intervals in culture. Furthermore, testosterone secretion was greater in DHG:F12 + Hepes + TIE than in M199-BSA at all time intervals tested. In DHG:F12 + Hepes + TIE, basal and gonadotropin-stimulated testosterone production by Leydig cells were maintained for 72 h in culture. Degenerative changes in morphology were apparent in some cells at 72 h, but not at earlier times in culture. This primary culture system for isolated Leydig cells provides a valuable tool to examine the temporally regulated events in Leydig cell function.     

Significant population variation in adult male height associated with the Y chromosome and the aromatase gene

The determination of human adult height is dependent on both environmental and genetic factors. Rare causes of abnormal stature have been identified, including mutations in the gene encoding aromatase (CYP19) and regions on the Y chromosome. However, the possible role of these loci in the genetic control of normal adult height is unknown. We have performed an association study using common biallelic polymorphisms within CYP19 and the Y chromosome to determine whether these loci are associated with variation in height in 413 adult males and 335 females drawn at random from a large population sample. An association between CYP19 and height was found (difference, 2.0 cm; 95% confidence interval, 0.16-3.8; P = 0.003), but this was more evident in men (difference, 2.3 cm; 95% confidence interval, 0.38-4.4; P = 0.05) than women (difference, 0.2 cm; 95% confidence interval, -2.1 to 1.6; P = 0.94). An association was also found with the Y chromosome (P = 0.009; difference of 1.9 cm; 95% confidence interval, 0.5-3.4). Additionally, when men were grouped according to haplotypes of the CYP19 and Y chromosome polymorphisms, a difference of 4.2 cm (95% confidence interval, 0.67-7.3) was detected (P = 0.004). These results suggest that in men, genetic variation in CYP19 and on the Y chromosome are involved in determining normal adult height, and that these loci may interact in an additive fashion.     

Epigenetic control of vasopressin expression is maintained by steroid hormones in the adult male rat brain

Although some DNA methylation patterns are altered by steroid hormone exposure in the developing brain, less is known about how changes in steroid hormone levels influence DNA methylation patterns in the adult brain. Steroid hormones act in the adult brain to regulate gene expression. Specifically, the expression of the socially relevant peptide vasopressin (AVP) within the bed nucleus of the stria terminalis (BST) of adult brain is dependent upon testosterone exposure. Castration dramatically reduces and testosterone replacement restores AVP expression within the BST. As decreases in mRNA expression are associated with increases in DNA promoter methylation, we explored the hypothesis that AVP expression in the adult brain is maintained through sustained epigenetic modifications of the AVP gene promoter. We find that castration of adult male rats resulted in decreased AVP mRNA expression and increased methylation of specific CpG sites within the AVP promoter in the BST. Similarly, castration significantly increased estrogen receptor α (ERα) mRNA expression and decreased ERα promoter methylation within the BST. These changes were prevented by testosterone replacement. This suggests that the DNA promoter methylation status of some steroid responsive genes in the adult brain is actively maintained by the presence of circulating steroid hormones. The maintenance of methylated or demethylated states of some genes in the adult brain by the presence of steroid hormones may play a role in the homeostatic regulation of behaviorally relevant systems.     

Temporal gene expression is restored concomitantly with germ cells in the experimentally regressed rat testis.

The present study was designed to examine the effect of hypophysectomy and subsequent testosterone administration on germ cell numbers and germ cell- and Sertoli cell-specific mRNA levels in adult rats. Rats were hypophysectomized and 4 weeks later received 24-cm testosterone-containing polydimethylsiloxane (PDS) implants. Sham-hypophysectomized rats received an empty PDS implant. At 0 and 3 days, and at 1, 2, 4, and 8 weeks, rats were killed. One testis from each rat (n = 4/group) was used to prepare total RNA; the other testis was used to enumerate stage VII-VIII germ cells. cDNA probes for germ cell and Sertoli cell products were used to monitor germ cell- and Sertoli cell-specific mRNAs on Northern blots. Four weeks after hypophysectomy (0 days), preleptotene and pachytene spermatocytes and round and elongating spermatids were reduced in number to 54%, 12%, 1%, and 0%, respectively, of the control values. Testosterone administration caused a time-dependent increase in germ cell numbers; after 8 weeks of testosterone treatment, preleptotene and pachytene spermatocytes and round and elongating spermatids were 75%, 79%, 74%, and 22%, respectively, of control values. Lactate dehydrogenase-C, phosphoglycerate kinase-2, protamine-1, and sulfated glycoprotein-2 mRNA levels (on a per micrograms RNA basis) were 34%, 34%, less than 1%, and 580% of control values, respectively, 4 weeks after hypophysectomy and 79%, 87%, 61%, and 192% of control values, respectively, after 8 weeks of testosterone treatment. Pachytene spermatocyte and round spermatid numbers increased, while Sertoli cell sulfated glycoprotein-2 mRNA levels decreased, with respect to 4 week hypophysectomy values, as early as 3 days after implantation of testosterone capsules. In contrast, germ cell (lactate dehydrogenase-C, phosphoglycerate kinase-2, and protamine-1) mRNA levels increased to the greatest extent between 1-4 weeks after the start of testosterone treatment and, after a short lag period, reflected increases in germ cell type and number. The results indicate that cell-specific mRNAs appear concomitantly with germ cell reappearance in a time-dependent manner in the testes of testosterone-treated hypophysectomized adult rats.     

A germ cell factor(s) modulates preproenkephalin gene expression in rat Sertoli cells.

Within the seminiferous tubule, both Sertoli and specific germ cells express opioid genes. Little is known about the paracrine regulation or role of opioid gene expression in the tubule. The present study shows that interactions among cells within the tubule may play a role in regulating preproenkephalin (PPenk) gene expression. Rat pachytene spermatocytes (PS) and round spermatids (RSd) were purified by centrifugal elutriation and established as primary cultures or co-cultured with Sertoli cells. The effects of germ cells or germ cell-conditioned media were studied to determine the expression of one of the opioid precursor genes in rat Sertoli cells, the PPenk gene. Following a 24 h co-culture with either PS or RSd, the expression of PPenk gene in Sertoli cells was increased 6.4- and 1.9-fold, respectively. Conditioned media obtained from either PS or RSd cultured for 20 h stimulated PPenk mRNA levels in Sertoli cells from as early as 2 h after exposure; maximum increases of 3.5- and 7.6-fold were observed at 12 h, respectively. The molecular weight of the germ cell factor(s) is greater than 30 kDa. 2 h after the addition of either PS- or RSd-conditioned media to Sertoli cells, small (2- to 2.6-fold, respectively) but significant (p less than 0.02) increases in extracellular cAMP levels were observed. Although both FSH and forskolin activated c-fos and PPenk gene expression in Sertoli cells, the germ cell factor(s) that stimulated PPenk mRNA levels did not affect the expression of this oncogene. These results indicate that germ cells interact with Sertoli cells, possibly by a protein(s) that acts as a short-loop paracrine factor, which regulates the expression of PPenk gene in Sertoli cells. These data suggest that stage-specific regulation of PPenk levels in Sertoli cells may occur in vivo.     

Gonadotropic regulation of aromatase activity in the adult rat testis

Aromatase activity during gonadotropin action in vivo and in vitro was examined in purified Leydig cells to further define the early effects of LH and for elucidation of the enzymatic processes involved in the development of late lesions of androgen biosynthetic pathway. Aromatase was measured by the tritiated water release method using [1 beta-3H]testosterone as substrate. The enzyme activity was proportional to the amount of cells (0.1-1.0 X 10(6] incubated, increased with the incubation time (2-60 min), was inhibited by androstatriendione (ED50, 5.0 microM), and showed a Km for testosterone of 1.69 microM. Aromatase activity was stimulated (10-20%; P less than 0.05) 1 h after treatment of rats with a single sc dose of 5 micrograms hCG. This activation preceded the late steroidogenic lesion at the site of 17 alpha-hydroxylase and 17,20-desmolase activity by 2-5 h. A RIA of improved sensitivity (0.5 pg) was developed to detect the very low cellular and secretory levels of estradiol. The testicular contents of testosterone and estradiol showed small increases (P less than 0.05) within 40 and 60 min after hCG treatment, respectively. Testicular testosterone levels reached a peak by 1 h after injection and preceded the peak level of estradiol formation by 2 h. After in vitro treatment of cultured Leydig cells with 100 ng hCG, the aromatase activity was significantly increased within 30 min (P less than 0.05) and then returned to control levels for up to 16 h of culture. A similar temporal pattern for enzyme activation was observed after treatment of cultures with 8-bromo-cAMP or forskolin (23-27% above control; P less than 0.05), while cholera toxin stimulated aromatase activity at 2 h. Net testosterone accumulation in the incubation medium increased 30 min after the hCG treatment and reached a plateau by 4 h. A small but significant increase in estradiol levels (P less than 0.05) was also observed at 30 min, remaining constant until 120 min, which was followed by a sharp rise parallel to that of testosterone. These results suggest that the estradiol-mediated desensitization of the Leydig cell observed after hCG administration is consistent with an early cAMP-dependent activation of aromatase and a further rise in estradiol formation due to increased substrate availability.     

Alteration in sex-specific behaviors in male mice lacking the aromatase gene

Brain aromatase (P450arom) is a key enzyme in estrogen biosynthesis from testicular androgens. This local aromatization in neural tissues is thought to be an important process for sexual differentiation and activation of sexual behavior in male rodents. To determine the functional significance of the aromatase gene in development and activation of sex-specific behavior, we analyzed a series of behavioral profiles in gonadally intact male mice with targeted disruption of exons 1 and 2 of the aromatase gene (ArKO). In most cases, ArKO males were infertile and showed deficits in male sexual behavior including mount, intromission and ejaculation. Noncontact penile erection was not significantly affected by deletion of the aromatase gene. A great reduction of aggressive behavior against male intruders was also observed in ArKO males, while they tended to exhibit aggression toward estrous females during male copulatory tests. Furthermore, 73% of ArKO males showed infanticide toward pups, whereas characteristic parental behavior, but not infanticide, was observed in wild-type males. These results support the brain aromatization hypothesis and indicate that aromatase gene expression is a critical step not only for motivational and consummatory aspects of male sexual behavior, but also for aggressive and parental behaviors in male mice.     

Preparation of thyrotroph cells from adult male rat pituitary glands by centrifugal elutriation

To study the metabolism of thyrotrophs and dynamics of TSH secretion in vitro, it is desirable to have a highly enriched population of thyrotrophs. For that purpose, centrifugal elutriation, a recently developed cell isolation method based on the size and density of cells, was used to prepare thyrotrophs from a cell suspension of adult male rat pituitary cells. Trypsin-dispersed cells (4-8 X 10(7] were loaded into the elutriation rotor (Beckman, JE-6) operating at 2800 rpm. Twelve cell fractions were collected at variable rotor speed (2000-2800 rpm) and increasing medium flow rate (10-103 ml/min). Cell recovery was 77-98%. The viability of the cells after elutriation was 90-95% based on trypan blue exclusion. Each fraction was analyzed for TSH, GH, and PRL content and for TRH-stimulated TSH release by RIA. Thyrotrophs were found predominantly in fractions 8-11 (flow rate 38-75 ml/min) based on TSH RIA. The mean TSH concentration in these fractions was 56 +/- 13.6 (+/- SD) microU/10(3) cells compared with 7.6 +/- 3.8 microU/10(3) cells in the initial cell suspension, representing a 7- to 8-fold enrichment of the thyrotrophs. Incubation with 20 nM TRH for 3 h increased the TSH release of cells eluted in fractions 8-11 by 3- to 5-fold; there was no significant increase in TSH release in fractions 3-6. Centrifugal elutriation may be used to prepare a uniform highly enriched thyrotroph fraction with excellent recovery from a suspension of rat pituitary cells. This technique should be valuable for study of the metabolism of thyrotrophs.     

Androgens and postmeiotic germ cells regulate claudin-11 expression in rat Sertoli cells

In the present study we investigated whether fetal exposure to flutamide affected messenger and protein levels of claudin-11, a key Sertoli cell factor in the establishment of the hemotesticular barrier, at the time of two key events of postnatal testis development: 1) before puberty (postnatal d 14) during the establishment of the hemotesticular barrier, and 2) at the adult age (postnatal d 90) at the time of full spermatogenesis. The data obtained show that claudin-11 expression was inhibited in prepubertal rat testes exposed in utero to 2 and 10 mg/kg x d flutamide. However, in adult testes, the inhibition was observed only with 2, and not with 10, mg/kg x d of the antiandrogen. It is shown here that these differences between prepubertal and adult testes could be related to dual and opposed regulation of claudin-11 expression resulting from positive control by androgens and an inhibitory effect of postmeiotic germ cells. Indeed, testosterone is shown to stimulate claudin-11 expression in cultured Sertoli cells in a dose- and time-dependent manner (maximum effect with 0.06 microm after 72 h of treatment). In contrast, postmeiotic germ cells potentially exert a negative effect on claudin-11 expression, because adult rat testes depleted in spermatids (after local irradiation) displayed increased claudin-11 expression, whereas in a model of cocultured Sertoli and germ cells, spermatids, but not spermatocytes, inhibited claudin-11 expression. The apparent absence of claudin-11 expression changes in adult rat testes exposed to 10 mg/kg x d flutamide therefore could result from the antagonistic effects of 1) the inhibitory action of the antiandrogen and 2) the stimulatory effect of the apoptotic germ cells on claudin-11 expression. Together, due to the key role of claudin-11 in the hemotesticular barrier, the present findings suggest that such regulatory mechanisms may potentially affect this barrier (re)modeling during spermatogenesis.     

Paracrine control of immature Sertoli cells by adult germ cells, in the rat (an in vitro study). Cell-cell interactions within the testis

Enriched populations of germ cells prepared from adult rats were found to influence 20-day-old rat Sertoli cell secretory activity by stimulating androgen-binding protein (ABP) and inhibiting oestradiol-17 beta production in the presence of follicle-stimulating hormone (FSH) as well as of dibutyryl cyclic AMP (dbcAMP). Among the different populations tested in coculture, pachytene spermatocytes were the most effective at stimulating ABP and inhibiting oestradiol production, whereas early spermatids had relatively less effects. Cytoplasts from elongated spermatids only slightly stimulated ABP secretion. The influence of germ cells upon Sertoli cells may be mediated via paracrine component(s) detected in nonconcentrated conditioned culture media. The stimulatory (ABP) and inhibitory (oestradiol) effects of pachytene spermatocyte and early spermatid-spent media were reversible (change of media), dose related, specific (no effect of cytoplast, peritubular cell, rat liver epithelial cell or 3T3 cell-conditioned media) and strictly proportional to the cell viability estimated at the end of the incubation periods. Furthermore, the nature of the germ cell factor(s) influencing Sertoli cell secretory function is likely to be proteinaceous since both germ cell-spent media effects were trypsin and heat (100 degrees C; 3 min) sensitive and retained by molecular weight (MW) greater than 10,000 cut-off dialysis membranes. It is hypothesized that germ cells, in particular pachytene spermatocytes and early spermatids, may influence Sertoli cell function during sexual development in the rat.