Martin Fritsch

Expression of the Insulin-Like Peptide 3 (INSL3) Hormone-Receptor (LGR8) System in the Testis

Abstract The new peptide hormone insulin-like peptide 3 (INSL3) is a member of the insulin-relaxin family, yet, unlike insulin, it signals through a new G-protein coupled receptor, LGR8, distantly related to the receptors for LH and FSH. INSL3 is produced in large amounts by the Leydig cells of the testis in both fetal and adult mammals. Using a combination of mRNA analysis by RT-PCR, immunohistochemistry, ligand-binding, and/or bioactivity assays, the distribution of LGR8 expression was assessed in testicular tissues and cells and in the epididymis. There was consistent agreement that LGR8 was expressed in meiotic and particularly postmeiotic germ cells and in Leydig cells, though not in Sertoli or peritubular cells. Leydig cells appear to express only a low level of the LGR8 gene product; other transcripts may be present, representing nonfunctional products. Messenger RNA analysis suggested that LGR8 transcripts in germ cells represented mostly full-length forms. LGR8 mRNA was also expressed in the epididymis, though no function can yet be ascribed to this expression. Therefore, the INSL3/LGR8 system represents a further paracrine hormone-receptor system in the testis, which conveys information about Leydig cell status to germ cells, and possibly as part of an autocrine feedback loop.

The sensitivity of murine spermiogenesis to miglustat is a quantitative trait: a pharmacogenetic study

BackgroundA major event in the post-meiotic development of male germ cells is the formation of the acrosome. This process can be perturbed in C57BL/6 mice by administration of the small molecule miglustat (N-butyldeoxynojirimycin, NB-DNJ). The miglustat-treated mice produce morphologically abnormal spermatozoa that lack acrosomes and are poorly motile. In C57BL/6 mice, miglustat can be used to maintain long-term reversible infertility. In contrast, when miglustat was evaluated in normal men, it did not affect spermatogenesis. To gain more insight into this species difference we have now evaluated the reproductive effects of miglustat in rabbits, in multiple mouse strains and in interstrain hybrid mice.MethodsMale mice of 18 inbred strains were administered miglustat orally or via miniosmotic pumps. Rabbits were given the compound in their food. Fourth-generation interstrain hybrid mice, bred from C57BL/6 and FVB/N mice (which differ in their response to miglustat), also received the drug. Data on fertility (natural mating), sperm motility and morphology, acrosome status, and serum drug levels were collected.ResultsIn rabbits the drug did not induce aberrations of sperm shape or motility, although the serum level of miglustat in rabbits far exceeded the level in C57BL/6 mice (8.4 μM and 0.5 μM, respectively). In some strains of the Swiss and Castle lineages of inbred mice miglustat did not cause infertility, severe morphological sperm aberrations or reduced sperm motility. In these strains miglustat only had milder effects. However, miglustat strongly disturbed acrosome and sperm nucleus development in AKR/J and BALB/c mice and in a number of C57BL/6-related strains. The consequences of drug administration in the interstrain hybrid mice were highly variable. Judging by the number of grossly abnormal spermatozoa, these genetically heterogeneous mice displayed a continuous range of intermediate responses, distinct from either of their parental strains.ConclusionThe effects of miglustat on spermatogenesis in mice are strain-dependent, while in rabbits the drug is ineffective. Evaluation of interstrain hybrid mice indicated that the sensitivity of spermatogenesis to miglustat is a quantitative trait. These studies pave the way for identifying the genetic factors underlying the strain/species differences in the effect of miglustat.

The expression of the testis-specific Dyrk4 kinase is highly restricted to step 8 spermatids but is not required for male fertility in mice

Members of the dual specificity tyrosine phosphorylated and regulated kinase family (Dyrk) were shown to have a highly testis-abundant or testis-restricted expression pattern. Furthermore, for some members of the family an involvement in gene expression regulation by phosphorylating transcription factors has been shown. Since little is known about the complex regulation of germ cell differentiation in spermatogenesis, we analysed the possible involvement of Dyrk kinases in this process. ISH experiments showed specific distribution of Dyrk kinases mainly in postmeiotic germ cell. We identified Dyrk4 as a testis-specific kinase with a very restricted expression to stage VIII postmeiotic spermatids. In vitro and in vivo experiments proved the enzymatic activity and suggested the cytoplasmatic localisation of Dyrk4. Finally, analysis of a Dyrk4 deficient mouse line showed that Dyrk4 is dispensable for male fertility, hence suggesting a functional redundancy of some Dyrk isoforms during spermiogenesis.

New approaches for male fertility control: HE6 as an example of a putative target.

Reversible contraceptive methods for males are still not available. During the last few years several marketing studies have clearly shown that men and women would welcome a situation where men could assume responsibility for family planning. Schering AG and Organon are currently collaborating to develop a hormonal method for male fertility control based on the combination of etonogestrel as gestagenic component and testosterone undecanoate. To further optimize male contraceptives in terms of improved efficiency, rapid onset, reversibility, fewer side effects and a convenient method of application, a search for innovative non-hormonal approaches was started. During the last few years, numerous proteins were identified which play a specific role in male fertility. These proteins have first to fulfil a set of indication-specific criteria before a drug discovery process can be initiated. The most important criteria for a putative target protein are tissue-selective expression, crucial biological function in fertility, drugable properties and feasibility of assay development for high-throughput-screening and lead optimization. The G-protein-coupled receptor HE6 was selected as target and the above selection criteria were applied. HE6 displays a preferred epididymis-specific expression pattern and belongs to the superfamily of GPCRs, which are well known to be drugable with small molecules. A knockout mouse was generated which revealed an infertility phenotype with the onset occurring 6 weeks after initiation of spermatogenesis at the latest. Surprisingly, no epididymis-specific phenotype was observed. Instead, the reabsorption of testicular fluid along the efferent ducts was strongly affected. No further obvious side effects were observed in male or female mice. This study with HE6 exemplifies how targets for male contraception have to be validated before drug development can start.

Post-meiotic gene products as targets for male contraception.

Post-meiotic stages of male germ cell maturation represent an interesting target system for the development of novel male contraceptive agents. In the human, these stages represent a period of only about 16 days differentiation, and thus targeting these cells would represent a contraceptive approach with a relatively rapid onset and equivalent recovery. Results from the Human Genome Project suggest that these cells also express a high number of very specific transcripts, though whether all of these are functional and/or essential for sperm differentiation and function requires more research. Until recently, however, these haploid stages were relatively inaccessible to molecular research because of the lack of appropriate model systems and methods. This situation has recently improved, with several new techniques involving manipulation of primary cells and seminiferous tubules, germ cell transplantation and the development of new immortalized cell-lines. Also, new biochemical approaches are yielding more information about haploid-specific transcription factors, such as GCNF. It is therefore to be expected that soon several new targets for a potential post-meiotic male contraceptive will become available for pharmaceutical development.