A new point mutation of the androgen receptor gene in a patient with partial androgen resistance and severe oligozoospermia.

Mutations of the androgen receptor gene in genetic males cause a variety of androgen insensitivity syndromes varying from female phenotype through intersexuality to male phenotype with infertility. The identification of a missense mutation in the steroid-binding domain in an infertile male with mild features of androgen insensitivity is reported here.

Stimulation of matrix-metalloproteinase-1 and tissue inhibitor of metalloproteinase-1 gene expression in rats by the preovulatory prolactin peak.

Since structural luteolysis involves deterioration of tissue, the gene expression of matrix-metalloproteinase-1 (MMP-1) and the respective tissue inhibitor of this metalloprotease (TIMP-1) were measured at various times on the day of pro-oestrus and in animals in which the preovulatory prolactin surge was blocked for the duration of 3 cycles by bromocriptine. An additional group of prolactin-blocked rats received a prolactin replacement injection on the afternoon of pro-oestrus. In spontaneously pro-oestrous rats, MMP-1 and TIMP-1 gene expression increased significantly (P<0.01) prior to the occurrence of the preovulatory LH surge but simultaneously with the onset of the preovulatory prolactin surge. When prolactin release was blocked by bromocriptine for 3 cycles, no such changes were observed during the afternoon of pro-oestrus. However, an intraperitoneal injection of bovine prolactin at the time when the preovulatory prolactin surge occurs normally, increased MMP-1 and TIMP-1 gene expression (P<0.01). These results indicate that MMP-1 and TIMP-1 gene expression are stimulated by the preovulatory prolactin surge. Previous work has shown that the preovulatory LH surge activates the enzymatic cascade which leads to increased collagenase activity.

Significance of the CAG repeat length in the androgen receptor gene (AR) for the transactivation function of an M780I mutant AR.

Mutations in the androgen receptor gene (AR) cause a wide spectrum of androgen insensitivity syndromes (AIS). Mutation analysis of patients with AIS has revealed that the same missense mutation of the AR gene can give rise to strongly divergent phenotypes suggesting the influence of modifying factors. The polymorphic CAG repeat in the first exon of the AR gene may be such a modifying factor. The influence of the length of the CAG repeat on the transactivation function of the M780I-mutant AR (causing partial and complete AIS) has been determined by cotransfection of HeLa cells with various CAG-AR expression vectors and a highly androgen-responsive luciferase reporter gene construct. The transcriptional activity of the M780I mutant AR can be, in contrast to the wild-type AR, considerably enhanced by non-physiologically high androgen concentrations. Furthermore, an inverse relationship between the number of the CAG repeats in the mutant AR and its activity has been observed.

Synergistic effects of prostaglandin F2alpha and tumor necrosis factor to induce luteolysis in the pig.

There is ample evidence that prostaglandin F2alpha (PGF2alpha) is a luteolytic substance in sows, however, there is also some evidence that it may stimulate progesterone (P4) secretion in young corpora lutea (CL). In vitro studies also suggested that tumor necrosis factor alpha (TNF) is inhibitory to luteal cell P4 and estradiol-17beta (E2) release. Since E2 is a strong luteotropic substance in porcine CL, we studied the effects of intraluteal application of PGF2alpha and TNF alone and in combination on the secretion of P4 and E2 in freely moving sows. Furthermore, the effects of intraluteal infusion of E2 and its stereoisomer, estradiol-17alpha, on luteal function, were also determined. Microdialysis systems were implanted into CL at Day 10 of the estrous cycle. After a 24-h recovery period, PGF2alpha (10(-6) M) or E2 (10(-6) M) was applied daily for 6 h into the CL. PGF2alpha caused a stimulation of E2 and P4, and E2 also stimulated P4 secretion at Days 11 and 12, but the stimulatory effect of both substances diminished as the CL approached luteolysis. Intraluteal TNF application resulted in a transient increase of P4 secretion, which was followed by a dramatic reduction of P4 release. When TNF-pretreated CL were exposed to PGF2alpha at Day 11 of the estrous cycle, the prostaglandin was no longer able to stimulate but rather inhibited E2 and P4 secretion. Intraluteal application of estradiol-17alpha had no effect on P4 secretion. These results are suggestive that the PGF2alpha-induced E2 secretion in young and middle-aged CL is stimulatory to P4 secretion. Under the influence of macrophage-derived TNF production, E2 secretion is inhibited, and thereby PGF2alpha and TNF cause functional luteolysis.

Clinical and molecular aspects of androgen receptor defects.

The androgen receptor (AR) is a ligand-dependent transcription factor involved in various biological processes such as sex differentiation, sexual maturation and spermatogenesis. Disorders of AR function cause a wide spectrum of androgen insensitivity syndromes. The phenotypes vary from women with female external genitalia through patients with genital ambiguity to men with normal male genitalia but infertile. The CAG repeat in exon A is important for transactivation function of the AR and consequently for many androgen-dependent processes. Expansion of this repeat is the cause of the X-linked spinal and bulbar muscular atrophy (SBMA, Kennedy's disease). Mutations of the AR gene occur commonly in prostate cancers and are significant for prognosis of the disease.

A(870)E mutation of the androgen receptor gene in a patient with complete androgen insensitivity syndrome and Sertoli cell tumor.

In a 60-year-old woman with complete androgen insensitivity syndrome (CAIS) and Sertoli cell tumor, a germline mutation (A870E) in exon 8 of the androgen receptor (AR) gene could be detected. A sister of the patient was also affected by CAIS and developed a Sertoli cell tumor at age 56. The mutation has not been described so far and could be seen in a causal relationship with the development of this tumor.

Immune-endocrine interactions affecting luteal function in pigs.

The formation, normal function and destruction of corpora lutea are essential features of normal reproduction. Although the formation of corpora lutea from follicles is largely dependent on pituitary gonadotrophins, the process of luteolysis is locally regulated and poorly understood. The corpus luteum consists of several steroidogenic and nonsteroidogenic cell types that interact with each other in a paracrine manner. Under cell culture conditions, large luteal cells that stem from follicular granulosa cells can be identified easily under the microscope and collected individually for single cell RT-PCR. As each of the 120 large luteal cells express the gene encoding 3 beta-hydroxysteroid dehydrogenase, it appears that all large luteal cells are steroidogenic. Large luteal cells also express the oestrogen receptor gene and as they are known to produce oestradiol, it can be concluded that the steroid acts in an auto- or intracrine manner in large luteal cells. Since we showed previously that oestradiol stimulates progesterone release under in vitro and in vivo conditions, it can be concluded that the steroid is an important intraluteally acting luteotrophic signal. At the time of luteal regression, macrophages invade the corpora lutea and their cytokine products, particularly tumour necrosis factor alpha (TNF alpha), appear to be involved in reduced steroid secretion. Indeed, TNF alpha inhibits production of progesterone and oestradiol from cultivated luteal cells. In sows, oestradiol is a strong luteotrophic factor and the production of oestradiol and of its receptor is downregulated by TNF alpha. Thereby, TNF alpha not only exerts direct luteolytic effects but also prevents the luteotrophic effects of oestradiol. Hence, it has an anti-luteotrophic action. In most species, functional luteolysis is accompanied by morphological regression of the corpus luteum. This structural luteolysis also appears to involve TNF alpha, as we have shown in pigs that expression of TNF alpha gene is high during luteolysis. Furthermore, TNF alpha stimulates programmed cell death (apoptosis) in luteal cells kept under culture conditions.

Luteotropic and luteolytic effects of oxytocin in the porcine corpus luteum.

The presence and the release of oxytocin (OT) by corpora lutea (CL) of a number of species (Wathes et al. 1986, Watkins and Choy 1988) including ruminants (Ivell and Richter 1984, Hirst et al. 1986, Rodgers et al. 1983, Sawyer et al. 1986), primates (Dawood and Khan-Dawood 1986, Khan-Dawood 1987, Maas et al. 1992, Khan-Dawood et al. 1993), and the pig (Pitzel et al. 1984, Einspanier et al. 1991, Jarry et al. 1992) have been amply verified. Conflicting results concerning the effects of OT on steroidogenesis have been published; the peptide has been shown to be luteotrophic (Sawyer et al. 1986, Maas et al. 1992, Jarry et al. 1990), to have no effects (Rodgers et al. 1985) or to be luteolytic (Auletta et al. 1984, Auletta et al. 1988, Pitzel et al. 1988) and it appears that this confusion is only in part due to species differences but also the age of the luteal tissue seems to be of crucial importance for the understanding of the effects of OT (Schams et al. 1983, Wuttke et al. 1993, 1994). In the present contribution we will focus largely on our results obtained in the pig and where applicable, compare them with those obtained in other species. We will thus demonstrate that OT is released by luteal cells (Jarry et al. 1990, Einspanier et al. 1991, Jarry et al. 1992) and that luteal cells have OT receptors (Sernia et al. 1989, Pitzel et al. 1993a) which mediate the effects of the peptide on steroidogenesis. Finally, we will address the question whether OT is inhibitory or stimulatory to progesterone (P) and estradiol (E2) release, and we will come to the conclusion that OT is both luteotropic and luteolytic (Wuttke et al. 1993, 1994). The CL of all species investigated so far consists of two steroidogenic cell types. The so-called large luteal cells stem from follicular granulosa cells and they appear to be barely responsive to luteinizing hormone (LH)/human chorionic gonadotrophin (hCG) but they are highly receptive to prostaglandin F2 alpha (PGF2 alpha) (Hansel and Dowd 1986, Pitzel et al. 1990). Furthermore, they appear to produce OT (Rodgers et al. 1983, Theodosis et al. 1986). The small luteal cells are believed to derive from the follicular theca cells (Hansel and Dowd 1986, Pitzel et al. 1990). They are LH-receptive but synthesize few, if any, regulatory peptides. In the last few years it has become increasingly evident that cells deriving from the white blood cell line are involved in processes such as ovulation and luteolysis. Of crucial importance for the understanding of luteolysis is the morphological observation that macrophages invade the CL at the time of luteal regression (Adashi 1990, Paavola 1977, Kirsch et al. 1981).

Porcine luteal cells express monocyte chemoattractant protein-2 (MCP-2): analysis by cDNA cloning and northern analysis.

From an expression library in lambda UniZAP, derived from porcine corpus luteum (CL), a clone lambda MCP9 was detected by hybridization with a porcine MCP-1 specific probe. A pBluescriptSK-derivative pMCP9 was generated from lambda MCP9 by in vivo excision and was shown to contain an open reading frame (ORF) encoding a protein highly homologous to bovine monocyte chemoattractant protein-2 (MCP-2). Comparison of amino acid sequences of known MCPs identified the protein encoded by pMCP9 as porcine MPC-2. The 3' untranslated region of pMCP9 was completed by 3' RACE. Northern analysis using RNA from porcine luteal cells and probes specific for porcine MCP-1 and MCP-2 revealed that porcine luteal cells express both MCPs. According to Southern analysis MCP-2, like MCP-1, is specified by a single copy gene.

Porcine luteal cells express monocyte chemoattractant protein-1 (MCP-1): analysis by polymerase chain reaction and cDNA cloning.

RT PCR employing poly(A+)RNA from porcine luteal cells and a combination of primers designed from the known bovine MCP-1 cDNA identified the luteal cells as a source of MCP-1. This finding is corroborated by results from Northern analysis using total RNA from luteal cells. To characterize the complete porcine MCP-1 cDNA, poly (A+)RNA was isolated from porcine corpus luteum, transcribed into cDNA and the latter cloned into the expression vector lambda Uni-ZapXR. A digoxigenin-labeled DNA probe of 375 bp was obtained by PCR and employed to screen the library. From the positive clones pMCP5, pMCP7 and pMCP10, the clone pMCP5 was selected and both strands of the cDNA insert were sequenced. The cDNA insert was 742 bp long, with an open reading frame (ORF) encoding a protein of 99 amino acid residues which by comparison with known amino acid sequences of MCPs yielded highest identities with MCP-1 sequences. We therefore assume that pMCP5 encodes the amino acid sequence for porcine MCP-1.

Luteotrophic and luteolytic actions of ovarian peptides.

Corpora lutea of all species investigated so far, including the human, produce oxytocin and a variety of other regulatory peptides. The role of these peptides is largely unknown. The subtypes of large luteal cells are able to produce tumour necrosis factor (TNF) and at the end of the luteal phase TNF-producing macrophages invade the aged corpus luteum, indicating that this cytokine may be involved in the process of luteolysis. The present contribution reviews briefly the known functions of oxytocin and substance P in the corpus luteum and then elaborates the possible involvement of luteal and macrophage TNF during luteolysis. Oxytocin applied to intact corpus luteum stimulates the secretion of progesterone and oestradiol. The stimulation of progesterone secretion by oxytocin is due to the stimulated oestrogen production. TNF, when tested in vitro, inhibits both luteal cell progesterone and oestradiol production. The TNF-mediated inhibition of aromatase activity therefore prevents the luteotrophic effects of a variety of peptides including oxytocin. This appears to be the mechanism by which TNF induces luteolysis.

Competitive PCR for quantitation of gonadotropin-releasing hormone mRNA level in a single micropunch of the rat preoptic area.

A competitive polymerase chain reaction (PCR) for quantitating gonadotropin-releasing hormone (GnRH) mRNA level in a single micropunch of the rat preoptic area (POA) is described. The POA (600 microns in depth) was micropunched from frozen rat brain slices and used for mRNA isolation using Dynabeads-oligo(dT) magnetic separation technique. The target RNA combined with a synthetic, deletion mutant GnRH cRNA as an internal standard, is co-reverse transcribed, and their cDNAs are subsequently co-amplified by Taq DNA polymerase in the same tube in which the same GnRH primers are used. This PCR protocol is sensitive enough to detect GnRH mRNA level in a single POA micropunch derived from an individual rat. There is a linear increase of the amount of GnRH PCR products as a function of input RNA and of the number of PCR cycles. Addition of mutant GnRH cRNA as an internal standard allows us to quantitate GnRH mRNA level in biological samples and to compensate variations of PCR reaction between samples. Following preoptic treatment with 5'-ADMP, which depletes selectively norepinephrine (NE), GnRH mRNA level was significantly reduced. This simple, yet highly sensitive PCR method appears to be a valuable tool for the study of the cellular and molecular regulation of GnRH gene expression in a variety of experimental models.