Biomedical subjects

A P Themmen

Publications and source records attributed to A P Themmen.

At least 19 recordsLinked to original sources

PubMed2005

Novel insertion frameshift mutation of the LH receptor gene: problematic clinical distinction of Leydig cell hypoplasia from enzyme defects primarily affecting testosterone biosynthesis.

Leydig cell hypoplasia (LCH) is a rare autosomal recessive condition that interferes with normal development of male external genitalia in 46,XY individuals and is caused by inactivating mutations of the LH receptor gene. The clinical and biochemical diagnostic parameters of LCH are not always specific and may therefore show significant overlap with other causes of insufficient testicular steroid biosynthesis. We have studied a 46,XY newborn with completely female external genitalia and palpable testes. Due to an increased basal serum ratio of androstenedione/testosterone, 17 beta-hydroxysteroid dehydrogenase type 3 (17 beta-HSD 3) deficiency was initially suspected. DNA analysis of the corresponding HSD17B3 gene, however, showed no abnormalities in the entire coding region. In contrast, direct sequencing of the LH receptor gene revealed a novel homozygous single nucleotide insertion in exon 11 (codon A589fs) producing a frame shift in the open reading frame predicting for premature termination of translation 17 amino acids downstream. From the genetic perspective, this mutation represents the first frame shift mutation in the LH receptor gene ever reported to date. From the clinical standpoint, LCH should always be considered in the differential diagnosis as steroid profiles may not be informative. Therefore, molecular genetic analysis should be warranted for androgen biosynthesis defects in all cases.

Amino Acid Sequence↗

PubMed2001

Lutropin receptor function: insights from natural, engineered, and computer-simulated mutations.

The lutropin receptor is a member of the superfamily of G protein-coupled receptors and, in humans, binds two closely related ligands, members of the heterodimer glycoprotein hormone family. As reviewed herein, comparative analyses of natural, engineered, and computer-simulated mutations of the lutropin receptor have provided considerable insight into the molecular basis of receptor function.

Amino Acid Sequence↗

PubMed2001

Anti-Müllerian hormone attenuates the effects of FSH on follicle development in the mouse ovary.

Although ovarian follicle growth is under the influence of many growth factors and hormones of which FSH remains one of the most prominent regulators. Therefore, factors affecting the sensitivity of ovarian follicles to FSH are also important for follicle growth. The aim of the present study was to investigate whether anti-Müllerian hormone (AMH) has an inhibitory effect on follicle growth by decreasing the sensitivity of ovarian follicles to FSH. Furthermore, the combined action of AMH and FSH on ovarian follicle development was examined. Three different experiments were performed. Using an in vitro follicle culture system it was shown that FSH-stimulated preantral follicle growth is attenuated in the presence of AMH. This observation was confirmed by an in vivo experiment showing that in immature AMH-deficient females, more follicles start to grow under the influence of exogenous FSH than in their wild-type littermates. In a third experiment, examination of the follicle population of 4-month-old wild-type, FSH beta-, AMH-, and AMH-/FSH beta-deficient females revealed that loss of FSH expression has no impact on the number of primordial and preantral follicles, but the loss of inhibitory action of AMH on the recruitment of primordial follicles in AMH-deficient mice is increased in the absence of FSH. In conclusion, these studies show that AMH inhibits FSH-stimulated follicle growth in the mouse, suggesting that AMH is one of the factors determining the sensitivity of ovarian follicles for FSH and that AMH is a dominant regulator of early follicle growth.

Animals↗

PubMed2001

The serine/threonine transmembrane receptor ALK2 mediates Müllerian inhibiting substance signaling.

Müllerian inhibiting substance (MIS or anti-Müllerian hormone) is a member of the transforming growth factor-beta family and plays a pivotal role in proper male sexual differentiation. Members of this family signal by the assembly of two related serine/threonine kinase receptors, referred to as type I or type II receptors, and downstream cytoplasmic Smad effector proteins. Although the MIS type II receptor (MISRII) has been identified, the identity of the type I receptor is unclear. Here we report that MIS activates a bone morphogenetic protein-like signaling pathway, which is solely dependent on the presence of the MISRII and bioactive MIS ligand. Among the multiple type I candidates tested, only ALK2 resulted in significant enhancement of the MIS signaling response. Furthermore, dominant-negative and antisense strategies showed that ALK2 is essential for MIS-induced signaling in two independent assays, the cellular Tlx-2 reporter gene assay and the Müllerian duct regression organ culture assay. In contrast, ALK6, the other candidate MIS type I receptor, was not required. Expression analyses revealed that ALK2 is present in all MIS target tissues including the mesenchyme surrounding the epithelial Müllerian duct. Collectively, we conclude that MIS employs a bone morphogenetic protein-like signaling pathway and uses ALK2 as its type I receptor. The use of this ubiquitously expressed type I receptor underscores the role of the MIS ligand and the MIS type II receptor in establishing the specificity of the MIS signaling cascade.

Activin Receptors, Type I↗

PubMed2001

Etiological studies of severe or familial hypospadias.

PURPOSE: Hypospadias is a congenital anomaly occurring in 1250 to 1830 live male births, of which 20% involve a severe type. The recurrence risk in families is high. In the majority of cases the underlying etiology remains unknown, which hampers further management based on the specific requirements associated with a specific etiology. MATERIALS AND METHODS: In a single center study 63 unselected cases of severe hypospadias were studied for all presently known causes of hypospadias using clinical as well as molecular biological techniques. Also, 16 families with hypospadias were analyzed for possible androgen receptor gene mutations. RESULTS: In 31% of cases of severe hypospadias the underlying etiology was identified. Of these 31% of cases 17% were due to complex genetic syndromes, 9.5% were due to chromosomal anomalies, and 1 involved the vanishing testes syndrome, the androgen insensitivity syndrome and 5alpha-reductase type 2 deficiency, respectively. Based on hormone stimulation tests Leydig cell hypoplasia and disorders of testosterone biosynthesis were suspected in some patients but not confirmed by mutation analysis of the respective genes. Familial hypospadias was due to androgen insensitivity in only 1 family but no other etiologies were identified in this group. CONCLUSIONS: Using patient history, physical examination, karyotyping, hormonal evaluation, including human chorionic gonadotropin testing in prepubertal cases and additional biochemical and molecular genetic evaluation, an etiological diagnosis was made in 31% of cases of severe hypospadias. This diagnosis has implications for further patient treatment. In addition, familial hypospadias is rarely due to the androgen insensitivity syndrome.

Adolescent↗

PubMed2000

Apoptotic and proliferative changes during induced atresia of pre-ovulatory follicles in the rat.

Atresia, a degenerative process through which many follicles are removed from the growing pool, involves apoptotic changes in the follicular granulosa cells. To identify histochemical markers of early stages of atresia, an in-vivo rat model was used which allowed the study of atresia of pre-ovulatory follicles in a synchronized and chronological order. By blocking the pre-ovulatory luteinizing hormone surge with a gonadotrophin-releasing hormone (GnRH) antagonist, ovulation of the pre-ovulatory follicles is prevented, after which these follicles became atretic. The first morphological sign of atresia (pyknotic granulosa cell nuclei) was found 27 h after injection of GnRH antagonist. Since the pre-ovulatory follicles gradually become atretic in a synchronous fashion, this model provided an opportunity to study and define markers of future atresia in pre-ovulatory follicles. Atresia involves apoptosis of granulosa cells, and therefore internucleosomal DNA fragmentation was examined. Using the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labelling (TUNEL) assay it was found that the first sign of internucleosomal DNA fragmentation in granulosa cells of pre-ovulatory follicles was detectable 24 h after GnRH antagonist treatment. In order to find an upstream marker of atresia, the 5-bromo-deoxyuridine (BrdU) labelling index was used as a measure of proliferation. Already at 14 h after GnRH antagonist treatment, when morphological signs of atresia were not yet present, a clear decrease in BrdU labelling index was found in the granulosa cells.

Animals↗

PubMed2000

Involvement of insulin-like factor 3 (Insl3) in diethylstilbestrol-induced cryptorchidism.

Recently, it has been shown that targeted inactivation of the Insl3 gene in male mice results in cryptorchidism. The Insl3 gene encodes insulin-like factor 3 (Insl3), which is expressed in fetal Leydig cells. The testicular factor Insl3 appears to play an important role in the transabdominal phase of testis descent, which involves development of the gubernaculum. Other studies have demonstrated that in utero exposure to diethylstilbestrol (DES), a synthetic estrogen, can lead to cryptorchidism both in humans and in animal models. The present study was undertaken to investigate whether prenatal DES-exposure might interfere with testicular Insl3 mRNA expression. Furthermore, the effect of DES on steroidogenic factor 1 (SF-1) mRNA expression level was determined, since it has been shown that SF-1 plays an essential role in transcriptional activation of the Insl3 gene promoter. Timed pregnant mice were treated with DES (100 microg/kg body weight) or vehicle alone on days E9 (gestational day 9) through E17. Control and DES-exposed mouse fetuses were collected at E16, E17 and E18, when transabdominal testis descent is taking place. Lack of gubernaculum development in DES-exposed animals was confirmed by histological analyses at E17. Expression of Insl3 and SF-1 mRNAs was studied in testes of control and DES-exposed fetuses at E16 and E18 by RNase protection assay. Prenatal DES-exposure resulted in a three-fold decrease in Insl3 mRNA expression level (P<0.005), at both E16 and E18. In contrast, DES treatment had no effect on the expression of SF-1 mRNA. These results support our hypothesis that DES may interfere with gubernaculum development by altering Insl3 mRNA expression, providing a possible mechanism by which DES may cause cryptorchidism.

Animals↗

PubMed1999

Control of primordial follicle recruitment by anti-Müllerian hormone in the mouse ovary.

The dimeric glycoprotein anti-Müllerian hormone (AMH) is a member of the transforming growth factor-beta superfamily of growth and differentiation factors. During male fetal sex differentiation, AMH is produced by Sertoli cells and induces degeneration of the Müllerian ducts, which form the anlagen of part of the internal female genital system. In females, AMH is produced by the ovary, but only postnatally. The function of AMH in the ovary is, however, still unknown. Female AMH null mice were reported to be fertile, with normal litter size, but this does not exclude a more subtle function for ovarian AMH. To investigate the function of AMH in the ovary, the complete follicle population was determined in AMH null mice, in mice heterozygous for the AMH null mutation, and in wild-type mice of different ages: 25 days, 4 months, and 13 months. In the present study we found that ovaries of 25-day- and 4-month-old AMH null females, compared to those of wild-type females, contain more preantral and small antral follicles. In addition, in 4- and 13-month-old AMH null females, smaller numbers of primordial follicles were found. Actually, in 13-month-old AMH null females, almost no primordial follicles could be detected, coinciding with a reduced number of preantral and small antral follicles in these females. In almost all females heterozygous for the AMH null mutation the number of follicles fell in between the numbers found in wild-type and AMH null females. In 4-month-old AMH null females serum inhibin levels were higher and FSH levels were lower compared to those in wild-type females. In contrast, inhibin levels were lower in 13-month-old AMH null females, and FSH levels were unchanged compared to those in wild-type females. Furthermore, the weight of the ovaries was twice as high in the 4-month-old AMH null females as in age-matched wild-type females. We conclude that AMH plays an important role in primordial follicle recruitment, such that more primordial follicles are recruited in AMH null mice than in wild-type mice; the mice heterozygous for the AMH null mutation take an in-between position. Consequently, the ovaries of AMH null females and those of females heterozygous for the AMH null mutation will show a relatively early depletion of their stock of primordial follicles. The female AMH null mouse may thus provide a useful model to study regulation of primordial follicle recruitment and the relation between follicular dynamics and ovarian aging.

Aging↗

PubMed1999

A limited repertoire of mutations of the luteinizing hormone (LH) receptor gene in familial and sporadic patients with male LH-independent precocious puberty.

Herein, we report mutation analysis of the LH receptor gene in 17 males with LH-independent precocious puberty, of which 8 were familial and 9 had a negative family history. A total of 7 different mutations (all previously reported) were detected in 12 patients. Among 10 European familial male-limited precocious puberty (FMPP) patients who had a LH receptor gene mutation, none had the Asp578Gly mutation, which is responsible for the vast majority of cases in the U.S. The restricted number of activating mutations of the LH receptor observed in this and other studies of FMPP strongly suggests that an activating phenotype is associated with very specific sites in the receptor protein. Clinical follow-up of the 5 patients who did not have LH receptor mutations shows that such cases most likely do not have true FMPP. LH receptor mutation analysis provides a sensitive tool for distinguishing true FMPP from other causes of early-onset LH-independent puberty in males.

Amino Acid Sequence↗

PubMed1999

Analysis of mutations in genes of the follicle-stimulating hormone receptor signaling pathway in ovarian granulosa cell tumors.

It has been suggested that ovarian granulosa cell tumors may result from unopposed hyperstimulation, either by excessive gonadotropin stimulation, by activating mutations of the FSH receptor gene, or of the G protein subunits, Gs alpha or Gi alpha2. We have examined the entire open reading frame of the FSH receptor gene in ovarian granulosa cell tumors. In addition, these tumors were evaluated for the known oncogenic G protein mutations Gsp and Gip2. Normal results were obtained in all 23 ovarian granulosa cell tumors. We conclude that mutations of the FSH receptor G protein signaling pathway do not play any major role in the genesis of these tumors.

Adult↗

PubMed1998

Downstream factors in transforming growth factor-beta family signaling.

The recently identified family of Smad proteins has given insight in the understanding of how members of the transforming growth factor-beta (TGF-beta) family relay their signal to the nucleus. Besides Smad proteins, G proteins and MAPKs are also involved in the downstream signaling of TGF-beta family members. The identification of elements that function downstream in the TGF-beta signaling pathway and the fact that these downstream players can interact with the signaling cascade of other growth factors, may give insight into the diverse biological responses evoked by the TGF-beta family members.

Calcium-Calmodulin-Dependent Protein Kinases↗

PubMed1998

Activating and inactivating mutations in LH receptors.

The glycoprotein hormones luteinizing hormone (LH) and human chorionic gonadotropin (hCG), both of which act through the LH receptor, play an important role in the regulation of both male and female gonadal function. Testicular androgen production by the Leydig cells is almost entirely dependent on LH/hCG action, while in the ovary LH triggers both oestrogen and progestin secretion and ovulation of the Graaffian follicles. In this review we discuss the various mutations that have been found in the LH receptor gene in the human and the lessons that can be learned from the phenotypes of the carriers of these mutations.

Female↗

PubMed1998

Isolation and characterization of all-trans-retinoic acid-responsive genes in the rat testis.

By way of differential screening of testis cDNA libraries from vitamin A-deficient (VAD) rats before and after administration of all-trans retinoic acid (ATRA), genes, the transcription of which was influenced by ATRA, were isolated. Most clones with an increased transcription encoded different subunits of the same mitochondrial protein complex, cytochrome c oxidase (COX). The mRNA expression of COX increased by a factor 3.9 +/- 1.5 (mean +/- SD, n = 4). This increased expression seems to reflect an increased energy demand in the ATRA-supplemented VAD testis. Also, one gene was isolated, the transcription of which was reduced to about 70% by ATRA. This gene, sulfated glycoprotein 2 (Sgp-2), is a major secretion product of Sertoli cells, the function of which is still unknown. The effect of ATRA on Sgp-2 expression may be direct, since the promoter of Sgp-2 contains a putative ATRA-responsive element (RARE).

Animals↗

PubMed1998

Differential regulation of leucine-rich primary response gene 1 (LRPR1) mRNA expression in rat testis and ovary.

In immature rat Sertoli cells, leucine-rich primary response gene 1 (LRPR1) represents a follicle stimulating hormone (FSH)-responsive gene; the function of the encoded protein is not yet known. LRPR1 mRNA expression is up-regulated very rapidly and specifically by FSH, both in cultured Sertoli cells and in vivo in regulation in more detail, in testis and ovary of fetal, immature, and adult rats. In addition, we have studied the expression of FSH receptor (FSHR) mRNA in relation to LRPR1 mRNA expression. In rat testis, LRPR1 mRNA and FSHR mRNA followed a similar expression pattern, during postnatal development and also at different stages of the spermatogenic cycle in the adult rat. Furthermore, after short-term challenge of the FSH signal transduction pathway in intact immature rats by injection with a relatively high dose of FSH, an inverse relationship between LRPR1 mRNA (up-regulation) and FSHR mRNA expression (down-regulation) was observed. Similar studies in the ovary provided completely different results. LRPR1 mRNA in the postnatal ovary is present well before expression of FSHR mRNA can be first detected. In addition, incubation of ovaries of immature rats with FSH or dibutyryl cyclic AMP (dbcAMP) did not result in up-regulation of LRPR1 mRNA expression. During fetal development, the LRPR1 mRNA expression pattern involved many more tissues, in contrast to the relatively tissue-specific expression of LRPR1 mRNA in gonads of 21 day old and adult rats. Moreover, LRPR1 mRNA expression could be detected as early as 12.5 days post-coitum, whereas FSHR mRNA is absent at this stage of fetal development. We concluded that the pronounced regulation of LRPR1 by FSH observed in the immature rat testis does not occur in the ovary. Furthermore, in the ovary LRPR1 mRNA expression does not appear to be dependent on FSH action. Finally, the LRPR1 gene product may play a general role during fetal development.

Animals↗

PubMed1998

Differential expression pattern of retinoid X receptors in adult murine testicular cells implies varying roles for these receptors in spermatogenesis.

Retinoids have previously been shown to be crucial for normal spermatogenesis. The role of retinoic acid receptors has been studied, but relatively little is known about the function of retinoid X receptors (RXRs). To gain more insight in the function of RXRs during spermatogenesis, the cellular localization of RXRs in the mouse testis was examined using immunohistochemistry and RNase protection assays. In both normal and vitamin A-deficient (VAD) testes, a strong immune response to an RXRalpha antibody occurred in Leydig cells, peritubular myoid cells, and A spermatogonia. Weaker signals were found in spermatocytes and spermatids. In normal testes, an RXRbeta antibody gave a reaction in Leydig cells, and, to a lesser extent, in Sertoli cells, A spermatogonia, pachytene spermatocytes, and spermatids. In Leydig cells, a cytoplasmatic signal was found in addition to the nuclear signal. In the VAD testis, only Leydig cells and A spermatogonia were positive, which indicates that RXRbeta expression may be dependent on the retinoid status. Previous studies have shown RXRgamma mRNA expression in the mouse testis at a low level. Nevertheless, an RXRgamma antibody caused a strong immune response in interstitial cells and in A spermatogonia, and a weak signal in pachytene spermatocytes. These immunohistochemical data were supported by the results of RNase protection assays on mRNA of testicular cell isolations. In conclusion, the different RXRs in the mouse testis have distinct expression patterns, suggesting that they may have different functions.

Animals↗

PubMed1998

Macroorchidism in FMR1 knockout mice is caused by increased Sertoli cell proliferation during testicular development.

The fragile X syndrome is the most frequent hereditary form of mental retardation. This X-linked disorder is, in most cases, caused by an unstable and expanding trinucleotide CGG repeat located in the 5'-untranslated region of the gene involved, the fragile X mental retardation 1 (FMR1) gene. Expansion of the CGG repeat to a length of more than 200 trinucleotides results in silencing of the FMR1 gene promoter and, thus, in an inactive gene. The clinical features of male fragile X patients include mental retardation, autistiform behavior, and characteristic facial features. In addition, macroorchidism is observed. To study the role of Sertoli cell proliferation and FSH signal transduction in the occurrence of macroorchidism in fragile X males, we made use of an animal model for the fragile X syndrome, an Fmr1 knockout mouse. The results indicate that in male Fmr1 knockout mice, the rate of Sertoli cell proliferation is increased from embryonic day 12 to 15 days postnatally. The onset and length of the period of Sertoli cell proliferation were not changed compared with those in the control males. Serum levels of FSH, FSH receptor messenger RNA expression, and short term effects of FSH on Sertoli cell function, as measured by down-regulation of FSH receptor messenger RNA, were not changed. We conclude that macroorchidism in Fmr1 knockout male mice is caused by an increased rate of Sertoli cell proliferation. This increase does not appear to be the result of a major change in FSH signal transduction in Fmr1 knockout mice.

Animals↗

PubMed1998

Effect of prenatal exposure to diethylstilbestrol on Müllerian duct development in fetal male mice.

The clinical use of diethylstilbestrol (DES) by pregnant women has resulted in an increased incidence of genital carcinoma in the daughters born from these pregnancies. Also, in the so-called DES-sons abnormalities were found, mainly, the presence of Müllerian duct remnants, which indicates that fetal exposure to DES may have an effect on male sex differentiation. Fetal regression of the Müllerian ducts is under testicular control through anti-Müllerian hormone (AMH). In male mice, treated in utero with DES, the Müllerian ducts do not regress completely, although DES-exposed testes do produce AMH. We hypothesized that incomplete regression in DES-exposed males is caused by a diminished sensitivity of the Müllerian ducts to AMH. Therefore, the effect of DES on temporal aspects of Müllerian duct regression and AMH type II receptor (AMHRII) messenger RNA (mRNA) expression in male mouse fetuses was studied. It was observed that Müllerian duct regression was incomplete at E19 (19 days post coitum), upon DES administration during pregnancy from E9 through E16. Furthermore, analysis of earlier time points of fetal development revealed that the DES treatment had clearly delayed the onset of Müllerian duct formation by approximately 2 days; in untreated fetuses, Müllerian duct formation was complete by E13, whereas fully formed Müllerian ducts were not observed in DES-treated male fetuses until E15. Using in situ hybridization, no change in the localization of AMH and AMHRII mRNA expression was observed in DES-exposed male fetuses. The mRNA expression was quantified using ribonuclease protection assay, showing an increased expression level of AMH and AMHRII mRNAs at E 13 in DES-exposed male fetuses. Furthermore, the mRNA expression levels of Hoxa 11 and steroidogenic factor-1 (SF-1) were determined as a marker for fetal development. Prenatal DES exposure had no effect on Hoxa 11 mRNA expression, indicating that DES did not exert an overall effect on the rate of fetal development. In DES-exposed male fetuses, SF-1 showed a similar increase in mRNA expression as AMH, in agreement with the observations that the AMH gene promoter requires an intact SF-1 DNA binding site for time- and cell-specific expression, although an effect of DES on SF-1 expression in other tissues, such as the adrenal and pituitary gland, cannot be excluded. However, the increased expression levels of AMH and AMHRII mRNAs do not directly explain the decreased sensitivity of the Müllerian ducts to AMH. Therefore, it is concluded that prenatal DES exposure of male mice delays the onset of Müllerian duct development, which may result in an asynchrony in the timing of Müllerian duct formation, with respect to the critical period of Müllerian duct regression, leading to persistence of Müllerian duct remnants in male mice.

Animals↗

PubMed1998

The effect of 9-cis-retinoic acid on proliferation and differentiation of a spermatogonia and retinoid receptor gene expression in the vitamin A-deficient mouse testis.

Retinoid X receptors (RXRs) are key regulators in retinoid signaling. Knowledge about the effects of 9-cis-retinoic acid (9-cis-RA), the natural ligand for the RXRs, may also provide insight in the functions of RXRs. In this study, the effect of 9-cis-RA on spermatogenesis in vitamin A-deficient (VAD) mice was examined. Administration of 9-cis-RA stimulated the differentiation and subsequent proliferation of the growth-arrested A spermatogonia in the testis of VAD mice. However, compared with all-trans-retinoic acid (ATRA), relatively higher doses of 9-cis-RA were necessary. This could not simply be due to a lower or delayed activity of 9-cis-RA, as simultaneous administration of ATRA and 9-cis-RA did not cause a synergistic effect. Instead, the presence of 9-cis-RA diminished the effect of ATRA by approximately one third. Studies of in vivo transport and metabolism showed that ATRA and 9-cis-RA, after administration to VAD mice, penetrated the testis equally well. However, 9-cis-RA was metabolized much faster than ATRA, and other metabolites were formed. This may account for the above-described differential effects of ATRA and 9-cis-RA on spermatogenesis. Similar to ATRA, 9-cis-RA transiently induced the messenger RNA expression of the nuclear RA receptor RAR beta, suggesting a role for this receptor in the effects of retinoids on the differentiation and proliferation of A spermatogonia. In contrast, the messenger RNA expression of the nuclear retinoid receptors RXR alpha, -beta, and -gamma was not changed significantly by administration of their ligand, 9-cis-RA. Hence, 9-cis-RA does not seem to exert its effect on spermatogenesis through altered expression of the RXRs.

Alitretinoin↗