Sequencing of a 'mouse azoospermia' gene panel in azoospermic men: identification of RNF212 and STAG3 mutations as novel genetic causes of meiotic arrest.

STUDY QUESTION: What is the diagnostic potential of next generation sequencing (NGS) based on a 'mouse azoospermia' gene panel in human non-obstructive azoospermia (NOA)? SUMMARY ANSWER: The diagnostic performance of sequencing a gene panel based on genes associated with mouse azoospermia was relatively successful in idiopathic NOA patients and allowed the discovery of two novel genes involved in NOA due to meiotic arrest. WHAT IS KNOWN ALREADY: NOA is a largely heterogeneous clinical entity, which includes different histological pictures. In a large proportion of NOA, the aetiology remains unknown (idiopathic NOA) and yet, unknown genetic factors are likely to play be involved. The mouse is the most broadly used mammalian model for studying human disease because of its usefulness for genetic manipulation and its genetic and physiological similarities to man. Mouse azoospermia models are available in the Mouse Genome Informatics database (MGI: http://www.informatics.jax.org/). STUDY DESIGN, SIZE, DURATION: The first step was to design of a 'mouse azoospermia' gene panel through the consultation of MGI. The second step was NGS analysis of 175 genes in a group of highly selected NOA patients (n = 33). The third step was characterization of the discovered gene defects in human testis tissue, through meiotic studies using surplus testicular biopsy material from the carriers of the RNF212 and STAG3 pathogenic variants. The final step was RNF212 and STAG3 expression analysis in a collection of testis biopsies. PARTICIPANTS/MATERIALS, SETTING, METHODS: From a total of 1300 infertile patients, 33 idiopathic NOA patients were analysed in this study, including 31 unrelated men and 2 brothers from a consanguineous family. The testis histology of the 31 unrelated NOA patients was as follows: 20 Sertoli cell-only syndrome (SCOS), 11 spermatogenic arrest (6 spermatogonial arrest and 5 spermatocytic arrest). The two brothers were affected by spermatocytic arrest. DNA extracted from blood was used for NGS on Illumina NextSeq500 platform. Generated sequence data was filtered for rare and potentially pathogenic variants. Functional studies in surplus testicular tissue from the carriers included the investigation of meiotic entry, XY body formation and metaphases by performing fluorescent immunohistochemical staining and immunocytochemistry. mRNA expression analysis through RT-qPCR of RNF212 and STAG3 was carried out in a collection of testis biopsies with different histology. MAIN RESULTS AND THE ROLE OF CHANCE: Our approach was relatively successful, leading to the genetic diagnosis of one sporadic NOA patient and two NOA brothers. This relatively high diagnostic performance is likely to be related to the stringent patient selection criteria i.e. all known causes of azoospermia were excluded and to the relatively high number of patients with rare testis histology (spermatocytic arrest). All three mutation carriers presented meiotic arrest, leading to the genetic diagnosis of three out of seven cases with this specific testicular phenotype. For the first time, we report biallelic variants in STAG3, in one sporadic patient, and a homozygous RNF212 variant, in the two brothers, as the genetic cause of NOA. Meiotic studies allowed the detection of the functional consequences of the mutations and provided information on the role of STAG3 and RNF212 in human male meiosis. LIMITATIONS, REASONS FOR CAUTION: All genes, with the exception of 5 out of 175, included in the panel cause azoospermia in mice only in the homozygous or hemizygous state. Consequently, apart from the five known dominant genes, heterozygous variants (except compound heterozygosity) in the remaining genes were not taken into consideration as causes of NOA. We identified the genetic cause in approximately half of the patients with spermatocytic arrest. The low number of analysed patients can be considered as a limitation, but it is a very rare testis phenotype. Due to the low frequency of this specific phenotype among infertile men, our finding may be considered of low clinical impact. However, at an individual level, it does have relevance for prognostic purposes prior testicular sperm extraction. WIDER IMPLICATIONS OF THE FINDINGS: Our study represents an additional step towards elucidating the genetic bases of early spermatogenic failure, since we discovered two new genes involved in human male meiotic arrest. We propose the inclusion of RNF212 and STAG3 in a future male infertility diagnostic gene panel. Based on the associated testis phenotype, the identification of pathogenic mutations in these genes also confers a negative predictive value for testicular sperm retrieval. Our meiotic studies provide novel insights into the role of these proteins in human male meiosis. Mutations in STAG3 were first described as a cause of female infertility and ovarian cancer, and Rnf212 knock out in mice leads to male and female infertility. Hence, our results stimulate further research on shared genetic factors causing infertility in both sexes and indicate that genetic counselling should involve not only male but also female relatives of NOA patients. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by the Spanish Ministry of Health Instituto Carlos III-FIS (grant number: FIS/FEDER-PI14/01250; PI17/01822) awarded to CK and AR-E, and by the European Commission, Reproductive Biology Early Research Training (REPROTRAIN, EU-FP7-PEOPLE-2011-ITN289880), awarded to CK, WB, and AE-M. The authors have no conflict of interest.

Targeted inactivation of mouse RAD52 reduces homologous recombination but not resistance to ionizing radiation.

The RAD52 epistasis group is required for recombinational repair of double-strand breaks (DSBs) and shows strong evolutionary conservation. In Saccharomyces cerevisiae, RAD52 is one of the key members in this pathway. Strains with mutations in this gene show strong hypersensitivity to DNA-damaging agents and defects in recombination. Inactivation of the mouse homologue of RAD52 in embryonic stem (ES) cells resulted in a reduced frequency of homologous recombination. Unlike the yeast Scrad52 mutant, MmRAD52(-/-) ES cells were not hypersensitive to agents that induce DSBs. MmRAD52 null mutant mice showed no abnormalities in viability, fertility, and the immune system. These results show that, as in S. cerevisiae, MmRAD52 is involved in recombination, although the repair of DNA damage is not affected upon inactivation, indicating that MmRAD52 may be involved in certain types of DSB repair processes and not in others. The effect of inactivating MmRAD52 suggests the presence of genes functionally related to MmRAD52, which can partly compensate for the absence of MmRad52 protein.

Characterization of a functional promoter for the Xenopus wnt-1 gene on vivo.

The Xenopus homolog of the proto-oncogene wnt-1 (int-1) is transiently expressed during neurula and tailbud stages of early development. To determine the mechanisms involved in the transcriptional regulation of the Xwnt-1 gene, we isolated Xwnt-1 genomic sequences. The promoter activity of the 5' flanking region of the gene was analysed by microinjection of chimeric luciferase reporter constructs into embryos. It is shown that the proximal 220 bp of the Xwnt-1 promoter is able to confer activation of the reporter gene at the early neurula stage. DNaseI footprinting analysis of the proximal promoter region with nuclear protein extracts from neurula stage embryos revealed five protein binding regions. Deletion and mutation analysis shows that one of these five protein binding regions is essential for promoter activity. Gel shift experiments indicate that a sequence element resembling the GT-I and GT-II motifs of the SV40 enhancer is important for the Xwnt-1 promoter activity in vivo.

Comparative analysis of Engrailed-1 and Wnt-1 expression in the developing central nervous system of Xenopus laevis.

Expression of the Engrailed-1 (XEn-1) gene was studied in Xenopus embryogenesis by Northern blot analysis and whole-mount in situ hybridization. One transcript of 2.2 kb was detected from stage 17 (midneurula) onwards, until stage 47 (swimming tadpole). The expression pattern of the XEn-1 gene as revealed by in situ hybridization can be divided in three regions. The first domain of transient expression appears at the midneurula stage (st. 17) in the anterior part of the neural fold, forming a complete ring of positive cells at the mid/hindbrain border after neural tube closure. A second region of transient expression is detected as groups of ventro-lateral cells in the spinal cord and the hindbrain from late-neurula till tadpole stages. A third area of transient expression of XEn-1 is formed by the anterior part of the developing pronephros. Comparison of XEn-1 expression at the mid/hindbrain border with that of the Xenopus wnt-1 and engrailed-2 genes reveals that XEn-1 and Xwnt-1, in contrast to XEn-2, are both detected in a narrow stripe of positive cells in this region. Analysis in exogastrulated embryos reveals that expression of XEn-1 and Xwnt-1, but not XEn-2, is induced by planar signaling in the presumptive midbrain. Of the three genes only XEn-1 is expressed in the floorplate at the mid/hindbrain border, while Xwnt-1 is expressed in adjacent cells in the neural ectoderm. The results suggest that in vertebrates at the interface between cells in the floorplate and in the paraxial neuroectoderm, at the limited region of the mid/hindbrain border, En-1 interacts with wnt-1 in a signaling pathway analogous to the engrailed/wingless signaling in the parasegments of the Drosophila embryo.

Anti-müllerian hormone and anti-müllerian hormone type II receptor messenger ribonucleic acid expression in rat ovaries during postnatal development, the estrous cycle, and gonadotropin-induced follicle growth.

During fetal development, anti-müllerian hormone (AMH) is produced only by Sertoli cells, but postnatally, granulosa cells also produce this peptide growth/differentiation factor. We recently identified a candidate AMH type II receptor (AMHRII). In the present study, postnatal ovarian AMH and AMHRII messenger RNA (mRNA) expression was studied by in situ hybridization and ribonuclease protection. In ovaries from adult rats, AMH and AMHRII mRNAs were found to be mainly expressed in granulosa cells from preantral and small antral follicles. Corpora lutea and large antral follicles express little or no AMH and AMHRII mRNA, and primordial follicles and oocytes appeared to be AMH and AMHRII mRNA negative. Thecal and interstitial cells express no detectable AMH mRNA and little or no AMHRII mRNA. The colocalization of AMH and AMHRII mRNAs in granulosa cells of specific follicle types suggests that actions of AMH via AMHRII are autocrine in nature. There is a decreased level of AMH and AMHRII mRNA expression when follicles become atretic. Both mRNA species are eventually lost from atretic follicles, although AMHRII mRNA expression seems to persist somewhat longer than AMH mRNA. During the estrous cycle, no marked changes in the patterns of AMH and AMHRII mRNA expression were detected, except at estrus, when expression of both mRNA species in preantral follicles was decreased compared to that on the other days of the cycle. On postnatal day 5, total ovarian AMH mRNA expression is low and is located in small preantral follicles. During the first weeks of postnatal development, AMH mRNA expression in preantral follicles increases, and the later formed small antral follicles also express AMH mRNA. In contrast, AMHRII mRNA is expressed on postnatal day 5 at a higher level than AMH mRNA, but cannot be localized to specific cell types. From postnatal day 15 onward, AMHRII mRNA expression becomes more restricted to the preantral and small antral follicles. Treatment of prepubertal rats with GnRH antagonist (Org 30276) and human recombinant FSH (Org 32489) or with GnRH antagonist and estradiol benzoate resulted in follicle growth and inhibition of AMH and AMHRII mRNA expression in some, but not all, preantral and small antral follicles. These results indicate that FSH and estrogens may play a role in the down-regulation of AMH and AMHRII mRNA expression in vivo when small antral follicles differentiate into large antral follicles. Furthermore, the FSH surge on the morning of estrus may inhibit AMH and AMHRII mRNA expression in preantral follicles.(ABSTRACT TRUNCATED AT 400 WORDS)

Anti-müllerian hormone and anti-müllerian hormone type II receptor messenger ribonucleic acid expression during postnatal testis development and in the adult testis of the rat.

Anti-müllerian hormone (AMH) induces degeneration of the müllerian ducts during male sex differentiation and may have additional functions concerning gonadal development. In the immature rat testis, there is a marked developmental increase in AMH type II receptor (AMHRII) messenger RNA (mRNA) expression in Sertoli cells, concomitant with the initiation of spermatogenesis. AMHRII mRNA is also expressed at a high level in Sertoli cells in adult rats. To obtain information about the possible functions of AMH in the testis, we investigated the postnatal expression patterns of the genes encoding AMH and AMHRII in the rat testis in more detail. Using RNase protection assays, AMH and AMHRII mRNA expression was measured in total RNA preparations from testes or testicular tubule segments isolated from control rats and from rats that had received various treatments. The testicular level of AMHRII mRNA was found to be much higher than that of AMH mRNA in adult rats. AMH mRNA was detected at a maximal level at stage VII of the spermatogenic cycle and at a low level at the other stages. AMHRII mRNA increases from stage XIII, is highest at stages VI and VII, and then rapidly declines at stage VIII to almost undetectable levels at stages IX-XII. It was found that the increase in testicular AMHRII mRNA expression during the first 3 weeks of postnatal development also occurs in sterile rats (prenatally irradiated), and hence, is independent of the presence or absence of germ cells. Yet, the total testicular level of AMHRII mRNA was decreased in sterile adult rats (prenatally irradiated or experimental cryptorchidism), as compared with intact control rats. However, treatment of adult rats with methoxyacetic acid or hydroxyurea, which resulted in partial germ cell depletion, had no effect on total testicular AMHRII mRNA expression. We conclude that a combination of multiple spermatogenic cycle events, possibly involving changes of Sertoli cell structure and/or Sertoli cell-basal membrane interactions, regulate autocrine AMH action on Sertoli cells, in particular at stage VII of the spermatogenic cycle.

Transient down-regulation of androgen receptor messenger ribonucleic acid (mRNA) expression in Sertoli cells by follicle-stimulating hormone is followed by up-regulation of androgen receptor mRNA and protein.

Cooperative actions of FSH and androgens on initiation, maintenance, and restoration of spermatogenesis have been described. In the present experiments the regulatory effects of FSH on androgen receptor (AR) gene expression in Sertoli cells were studied. In immature rats injection of FSH (1 microgram/g BW, ip) resulted in a rapid down-regulation of testicular AR mRNA expression (4 h), followed by recovery to the control level (10 h). Using cultured immature Sertoli cells, a similar transient effect on AR mRNA expression was observed after the addition of FSH (500 ng/ml) or (Bu)2cAMP (0.5 mM). Cycloheximide treatment of the cells did not prevent the rapid FSH-induced down-regulation of AR mRNA expression, indicating that de novo protein synthesis is not required for this effect. Furthermore, using a transcriptional run-on assay, no marked decrease in the rate of AR gene transcription was found upon treatment of the cultured Sertoli cells with FSH for 2 or 4 h. This demonstrates that the short term effect of FSH or AR mRNA expression reflects a change in mRNA stability. The AR protein level was not markedly affected by the transient decrease in AR mRNA expression. When immature Sertoli cells were incubated with FSH for longer time periods (24-72 h), both AR mRNA and protein expression were increased. In Sertoli cells isolated from 15-day-old rats, this increase was higher (mRNA, 2- to 3-fold; protein, 2-fold) than in Sertoli cells isolated from 25-day-old animals. The results indicate that FSH plays a complex role in the regulation of AR expression in immature rat Sertoli cells.

Chromatin dynamics in the male meiotic prophase.

During the male meiotic prophase in mouse and man, pairing and recombination of homologous chromosomes is accompanied by changes in chromatin structure. In this review, the dynamics of assembly and disassembly of the chromatin-associated complexes that mediate sister chromatid cohesion (cohesin) and maintain chromosome pairing (the synaptonemal complex) are described. Special features of the meiotic S phase are discussed, and also the dynamics of several key players that act together after the S phase at sites of meiotic double-strand break DNA repair. Current knowledge on histone modifications that occur during the male meiotic prophase is discussed, with special attention for the inactive chromatin of the X and Y chromosomes that constitutes the sex body. Finally, it is discussed that in the future, it will be possible to view the true chromatin dynamics during male meiosis in time, in living cells, through analysis of fluorescent-tagged proteins expressed in transgenic mice, using advanced fluorescent microscopy techniques.

A cytochemical staining procedure for succinate dehydrogenase activity in pre-ovulatory mouse oocytes embedded in low gelling temperature agarose.

We describe a cytochemical staining procedure for succinate dehydrogenase (SDH) activity in pre-ovulatory mouse oocytes. The oocytes were embedded in low gelling temperature agarose and treated with caffeine before cytochemical staining in the presence of nitro blue tetrazolium (NBT), phenazinemethosulfate (PMS), and succinate. This resulted in intense staining of the oocytes by formazan precipitate. The level of aspecific formazan production in the absence of succinate was very low. We applied the procedure to oocytes matured in vitro and found that the location of the formazan precipitate as a result of SDH activity correlated well with the location of mitochondria. The chromatin of the cytochemically stained oocytes could subsequently be analyzed by means of the DNA-specific fluorochrome DAPI. In pre-ovulatory oocytes, we found a correlation between chromatin organization and the location of mitochondria: in oocytes with an intact germinal vesicle the mitochondria were uniformly distributed in the cytoplasm, as shown by fine grains of formazan precipitate. In oocytes with condensed chromatin the mitochondria apparently had clustered, because the formazan precipitate was more coarse in these cells.

Welcome to the family: the anti-müllerian hormone receptor.

Anti-müllerian hormone (AMH) is a member of the superfamily of peptide growth/differentiation factors which includes the activins and TGF-beta s. The putative AMH type II receptor, which was cloned recently (Baarends et al., 1994), is a member of the superfamily of transmembrane serine/threonine kinase receptors. In hypothetical evolutionary relationship dendrograms, both AMH and its putative receptor take isolated positions relative to their respective family members. The prenatal expression pattern of this putative AMH receptor is in accordance with the expected endocrine action of AMH on the mesenchymal cells located adjacent to the müllerian duct, and with known effects of AMH on gonadal differentiation. Postnatal expression of mRNA encoding this receptor in granulosa and Sertoli cells provides a new stimulus to study possible functions of AMH in the gonads.

The ubiquitin system in gametogenesis.

Ubiquitin is a ubiquitous and highly conserved protein of 76 amino acid residues, that can be covalently attached to cellular acceptor proteins. The attachment of ubiquitin to target proteins is achieved through a multi-step enzymatic pathway, which involves activities of ubiquitin-activating E1 enzymes, ubiquitin-conjugating E2 enzymes, and ligating E3 enzymes. Mono- or poly-ubiquitination of proteins can lead to protein degradation or modification of protein activity. Many components of the complex ubiquitin system show remarkable evolutionary conservation, from yeast to mammalian species. The ubiquitin system is essential to all eukaryotic cells. Among others, several signal transduction cascades show involvement of the ubiquitin system, but there are currently little data supporting a specific role of the ubiquitin system in hormonal control of reproduction. Interestingly, during gametogenesis, many specialized and important aspects of the ubiquitin system become apparent. Components of the ubiquitin system appear to be involved in different steps and processes during gametogenesis, including control of meiosis, and reorganization of chromatin structure.

Regulation of gene expression in Sertoli cells by follicle-stimulating hormone (FSH): cloning and characterization of LRPR1, a primary response gene encoding a leucine-rich protein.

Searching for hormone-regulated genes in testicular Sertoli cells, we cloned and sequenced a cDNA of 3108 base pairs, named LRPR1 (signifying leucine-rich primary response gene 1). This cDNA sequence has an open reading frame of 2238 base pairs encoding a leucine-rich protein of 746 amino acid residues with a relative molecular mass of 85.6 kDa. As much as 16% of the amino acid residues is leucine. Database analysis revealed significant similarity of LRPR1 to the human brain cDNA sequence EST00443, but not to any other sequences present in databases. The expression of LRPR1 mRNA in Sertoli cells is strongly and rapidly up-regulated by follicle-stimulating hormone (FSH). The level of LRPR1 mRNA was very low in Sertoli cells isolated from 21-day-old rats and cultured for 3 days in the absence of FSH, but LRPR1 mRNA expression was markedly increased within 2 h after addition of FSH to these cultures. A maximal response was reached within 4 h. Dibutyryl-cyclic AMP [(Bu)2cAMP] and forskolin had similar effects compared to FSH, indicating that cAMP acts as a second messenger in the regulation of LRPR1 expression. The up-regulation of LRPR1 mRNA expression by FSH was also observed in the presence of the protein synthesis inhibitor cycloheximide, indicating that FSH regulates LRPR1 mRNA expression through a direct mechanism which does not require de novo protein synthesis. Thus, LRPR1 represents a primary response gene in FSH action on Sertoli cells. The presently available data indicate that LRPR1 mRNA expression is regulated specifically by FSH, since several other hormones and growth factors did not affect LRPR1 mRNA expression in the cultured Sertoli cells. LRPR1 mRNA expression is relatively high in testis, ovary and spleen. A much lower mRNA level was found in brain and lung, and no expression was detected in liver, kidney, heart, muscle, pituitary gland, prostate, epididymis and seminal vesicle. The basal level of testicular LRPR1 expression in intact 21-day-old rats was markedly increased within several hours after a single i.p. injection of FSH, indicating that in vivo LRPR1 mRNA expression may appear to be a useful parameter to evaluate testicular FSH action.

Knockout mouse model and gametogenic failure.

To evaluate the function of a defined gene in gametogenesis, exciting opportunities are offered by the introduction of techniques to generate knockout mice. In this short article, we briefly describe a few gene knockout mouse models, which show a phenotype that involves impairment of gametogenesis and/or fertility. The focus will be on the mHR6B gene knockout mouse, which shows male infertility. The mHR6B gene encodes an ubiquitin-conjugating enzyme, and the data point to an important role of the ubiquitin pathway in gametogenesis.

Follitropin receptor down-regulation involves a cAMP-dependent post-transcriptional decrease of receptor mRNA expression.

The regulation by FSH (follitropin; follicle-stimulating hormone) of FSH receptor mRNA and protein (FSH binding) was studied using cultured Sertoli cells isolated from 21-day-old rats. FSH induced a dose-dependent and almost complete down-regulation of receptor mRNA at 4 h after addition of the hormone. At subsequent time points (16 h and later) the FSH receptor mRNA levels had returned close to control values. The effect of FSH was mimicked by dibutyryl cyclic AMP (dbcAMP) and forskolin, and the phosphodiesterase inhibitor methyl-isobutylxanthine (MIX) prolonged the FSH action. These findings indicate that the effect of FSH on its receptor mRNA was mediated by cAMP. A down-regulatory effect of FSH and dbcAMP on FSH receptor mRNA was also observed in the presence of the protein synthesis inhibitor cycloheximide, suggesting a direct effect of FSH/dbcAMP on the expression of the FSH receptor gene. Transcriptional run-on experiments revealed that FSH did not inhibit initiation of the FSH receptor gene; hence a post-transcriptional mechanism is involved. Binding of 125I-FSH to the cultured Sertoli cells was rapidly (4 h) decreased when the cells were incubated with FSH or FSH in combination with MIX. This effect can be explained by ligand-induced receptor sequestration. In contrast, incubation of Sertoli cells with dbcAMP had no effect on binding of 125I-FSH after 4 h, but resulted in a 60% loss of FSH binding sites after 24 h, probably caused by decreased mRNA expression. In conclusion, FSH receptor down-regulation in Sertoli cells is effected not only by the well-documented ligand-induced loss of receptors from the plasma membrane, but also involves a cAMP-mediated decrease of FSH receptor mRNA through a post-transcriptional mechanism.

Transcriptional regulation of androgen receptor gene expression in Sertoli cells and other cell types.

Regulation of androgen receptor (AR) mRNA expression was studied in Sertoli cells and peritubular myoid cells isolated from immature rat testis, and in the lymph node carcinoma cell line derived from a human prostate (LNCaP). Addition of dibutyryl-cyclic AMP (dbcAMP) to Sertoli cell cultures resulted in a rapid transient decrease in AR mRNA expression (5 h), which was followed by a gradual increase in AR mRNA expression (24-72 h). This effect of dbcAMP mimicked follicle-stimulating hormone (FSH) action. In peritubular myoid cells, there was only a moderate but prolonged decrease during incubation in the presence of dbcAMP, and in LNCaP cells no effect of dbcAMP on AR mRNA expression was observed. When Sertoli cells or peritubular myoid cells were cultured in the presence of androgens, AR mRNA expression in these cell types did not change. This is in contrast to LNCaP cells, that showed a marked reduction of AR mRNA expression during androgen treatment. In the present experiments, transcriptional regulation of AR gene expression in Sertoli cells and LNCaP cells was also examined. Freshly isolated Sertoli cell clusters were transfected with a series of luciferase reporter gene constructs, driven by the AR promoter. It was found that addition of dbcAMP to the transfected Sertoli cells resulted in a small but consistent increase in reporter gene expression (which was interpreted as resulting from AR promoter activity); a construct that only contained the AR 5' untranslated region of the cDNA sequence did not show such a regulation. The same constructs, transfected into LNCaP cells, did not show any transcriptional down-regulation when the synthetic androgen R1881 was added to the cell cultures. A nuclear transcription elongation experiment (run-on), however, demonstrated that androgen-induced AR mRNA down-regulation in LNCaP cells resulted from an inhibition of AR gene transcription. The present results indicate that in Sertoli cells and LNCaP cells, hormonal effects on AR gene transcription play a role in regulation of AR expression. However, AR gene transcription in these cells is differentially regulated.

The rat androgen receptor gene promoter.

The androgen receptor (AR) is activated upon binding of testosterone or dihydrotestosterone and exerts regulatory effects on gene expression in androgen target cells. To study transcriptional regulation of the rat AR gene itself, the 5' genomic region of this gene was cloned from a genomic library and the promoter was identified. S1-nuclease protection analysis showed two major transcription start sites, located between 1010 and 1023 bp upstream from the translation initiation codon. The area surrounding these start sites was cloned in both orientations in a CAT reporter plasmid. Upon transfection of the constructs into COS cells, part of the promoter stimulated transcription in an orientation-independent manner, but the full promoter showed a higher and unidirectional activity. In the promoter/reporter gene constructs, transcription initiated from the same positions as in the native gene. Sequence analysis showed that the promoter of the rat AR gene lacks typical TATA and CCAAT box elements, but one SP1 site is located at about 60 bp upstream from the major start site of transcription. Other possible promoter elements are TGTYCT sequences at positions -174 to -179, -434 to -439., -466 to -471, and -500 to -505, resembling half-sites of the glucocorticoid-responsive element (GRE). Furthermore, a homopurine stretch containing a total of 8 GGGGA elements and similar to sequences that are present in several other GC-rich promoters, is located between -89 and -146 bp upstream from the major start site of transcription.

Follicle-stimulating hormone and testosterone stimulation of immature and mature Sertoli cells in vitro: inhibin and N-cadherin levels and round spermatid binding.

The in vitro response of Sertoli cells isolated from adult rat testes to testosterone (T) and follicle-stimulating hormone (FSH) treatment was investigated. Sertoli cells from >70-day-old Sprague-Dawley rats were isolated by a combined enzymatic treatment followed by the removal of the majority of contaminating germ cells with immobilized peanut agglutinin lectin. Sertoli cells were then cultured for 6-10 days, forming a confluent layer with a cell viability of >83% and 74-77% purity. The contaminating cells were peritubular cells (4-6%), pachytene spermatocytes (4-5%), round spermatids (<2%), elongated spermatids (<1%), and degenerating germ cells (14.8%). The proportion of degenerating germ cells decreased from 14.8% to 8.6% between days 6 and 10 in culture. After a prestimulation culture period of 4 days, FSH treatment over a 2-day period resulted in a dose-related increase of inhibin with a median effective dose (ED50) value of 36.7+/-20.4 ng/ml in comparison with an ED50 value of 4.4+/-0.9 ng/ml obtained with immature Sertoli cell cultures from 20-day-old rats. Mature Sertoli cells, in contrast to immature Sertoli cells, were unresponsive to combined FSH + T treatment for the production of the cell adhesion protein N-cadherin. FSH treatment promoted the in vitro binding of round spermatids isolated from adult testis to adult Sertoli cells in coculture. It is concluded that mature Sertoli cells in culture are responsive to FSH in terms of inhibin production and round-spermatid binding. The lack of an FSH + T-induced increase in N-cadherin or round spermatid binding is attributed to either a reduced sensitivity, or an alteration in the regulation of mature Sertoli cells by FSH + T.

Evolution of testis-specific kinases TSSK1B and TSSK2 in primates.

The testis-specific serine/threonine protein kinases TSSK1 and TSSK2 are known to be essential for male fertility, in mice. The enzymes are present in elongating spermatids, and targeted deletion of the two genes Tssk1 and Tssk2 results in dysregulation of spermiogenesis. The mouse genes are genetically closely linked, forming a Tssk1-Tssk2 tandem. In human, TSSK1 is present in the form of a pseudogene, TSSK1A, which is linked to an intact TSSK2 gene, and in the form of an intact gene, TSSK1B, which is not genetically linked to TSSK2. Studies on conservation of genes and gene function between mouse and human are relevant, to be able to use mouse models for studies on human infertility, and to evaluate possible targets for non-hormonal contraception targeting the male. Therefore, we have performed a detailed analysis of the evolution of genes encoding TSSK1 and TSSK2 among mammals, in particular among primates. This study includes functional analysis of replacement mutation K27R in TSSK2, which is frequently observed among humans. In primates, the kinase domains of TSSK1B and TSSK2 have evolved under negative selection, reflecting the importance to maintain their kinase activity. Positive selection was observed for the C-terminal domain of TSSK1B, which indicates that TSSK1B and TSSK2 may perform at least partly differential functions.

Mutations that affect meiosis in male mice influence the dynamics of the mid-preleptotene and bouquet stages.

Meiosis pairs and segregates homologous chromosomes and thereby forms haploid germ cells to compensate the genome doubling at fertilization. Homologue pairing in many eukaryotic species depends on formation of DNA double strand breaks (DSBs) during early prophase I when telomeres begin to cluster at the nuclear periphery (bouquet stage). By fluorescence in situ hybridization criteria, we observe that mid-preleptotene and bouquet stage frequencies are altered in male mice deficient for proteins required for recombination, ubiquitin conjugation and telomere length control. The generally low frequencies of mid-preleptotene spermatocytes were significantly increased in male mice lacking recombination proteins SPO11, MEI1, MLH1, KU80, ubiquitin conjugating enzyme HR6B, and in mice with only one copy of the telomere length regulator Terf1. The bouquet stage was significantly enriched in Atm(-/-), Spo11(-/-), Mei1(m1Jcs/m1Jcs), Mlh1(-/-), Terf1(+/-) and Hr6b(-/-) spermatogenesis, but not in mice lacking recombination proteins DMC1 and HOP2, the non-homologous end-joining DNA repair factor KU80 and the ATM downstream effector GADD45a. Mice defective in spermiogenesis (Tnp1(-/-), Gmcl1(-/-), Asm(-/-)) showed wild-type mid-preleptotene and bouquet frequencies. A low frequency of bouquet spermatocytes in Spo11(-/-)Atm(-/-) spermatogenesis suggests that DSBs contribute to the Atm(-/-)-correlated bouquet stage exit defect. Insignificant changes of bouquet frequencies in mice with defects in early stages of DSB repair (Dmc1(-/-), Hop2(-/-)) suggest that there is an ATM-specific influence on bouquet stage duration. Altogether, it appears that several pathways influence telomere dynamics in mammalian meiosis.