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Laboratory for Experimental Medicine and Endocrinology Catholic University of Leuven B-3000 Leuven, Belgium
Address all correspondence and requests for reprints to: Guido Verhoeven, Professor, Catholic University of Leuven, Laboratory for Experimental Medicine and Endocrinology, Department of Experimental Medicine, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium. E-mail: guido.verhoeven{at}med.kuleuven.be.
The gonadotropins LH and FSH act as the master switches that control both development and function of the adult testis (1). Research over the past 25 yr has overtly shown, however, that they are by far not the only regulatory molecules involved in the control of testicular function and, more in particular, in the control of spermatogenesis. Adequate proliferation of spermatogonia, meiotic progression of spermatocytes, differentiation of spermatids, and release of spermatozoa depend on a hierarchical and coordinated control system comprising, besides gonadotropins, a complex intratesticular network of cell-to-cell interactions providing developing germ cells with the appropriate environment and the essential cues to express their built-in genetic program (2, 3, 4, 5, 6). The pivotal role of this intratesticular network of communications is underlined by the fact that neither FSH nor LH act directly on the germ cells. FSH receptors are found only on Sertoli cells (7), the somatic cells that support developing germ cells throughout their journey to the center of the seminiferous tubules, whereas LH acts on the Leydig cells in the interstitial compartment to provide high intratesticular levels of androgens that again use Sertoli cells as their primary targets (8, 9).
Cell-to-cell interactions in the testis may take different forms and have been classified as environmental, nutritional, or regulatory (2). Extracellular matrix elements and cell adhesion molecules play an important role in the establishment of environmental interactions. They contribute for instance to the polarization of Sertoli cells and to the formation of basally located specific tight junctions between adjacent Sertoli cells that form the Sertoli cell barrier (also referred to as the blood-testis barrier). This barrier divides the tubules into two functional compartments: a basal compartment containing mainly spermatogonia and an adluminal compartment providing the appropriate environment for all further stages of germ cell development (10). Nutritional interactions between Sertoli cells and germ cells foster germ cell development by providing adequate substrates, minerals, and vitamins within this adluminal compartment. Regulatory interactions involve paracrine and autocrine signaling molecules mediating, modulating, and coordinating the effects of the gonadotropins.
More than 30 growth factors, cytokines, and hormones have been suggested to act as local regulatory agents in the testis (2, 3, 4, 5, 6). For all these factors, a variable amount of evidence has been accumulated indicating that they are produced by somatic or germinal cells in the testis, that cognate receptors are expressed by testicular cells, and that they are able to exert biological effects on one or more testicular target cells. Regulatory factors have been suggested to be involved in the multidirectional communications between Sertoli cells, germ cells, Leydig cells, and peritubular myoid cells (2, 3, 4). The unraveling of the exact networks involved, however, remains a major challenge. The main part of our present understanding of regulatory interactions in the testis relies on in vitro observations in isolated cell types. Attempts to clarify the in vivo significance of putative regulatory factors by transgenic approaches involving targeted gene inactivation or overexpression have been limited and interpretation has been hampered by a variety of problems such as occurrence of lethal phenotypes, functional redundancies within the system, activation of adaptive mechanisms, and extratesticular effects of the targeted genes (4).
In this issue of Endocrinology, Usdin et al. (11) describe a quite unique experimental paradigm in which inactivation of a gene encoding tuberoinfundibular peptide of 39 residues (TIP39), a candidate local regulatory factor in the testis, results in a complete block in meiosis and in which fertility can be rescued in TIP39-defective (Tifp39–/–) mice by restoring its expression selectively in germ cells. TIP39 was originally purified from bovine hypothalamus and sequenced in 1999 (12). It was identified as a specific ligand for the PTH2 receptor with a suggested role in the control of the secretion of hypothalamic-releasing factors and in nociception (13, 14). From the very beginning, however, it was clear that the neuromodulator TIP39 might also have actions outside the brain. Both Northern blot analysis and RT-PCR point to TIP39 expression in the testis and also in the eye (13, 15). In situ hybridization in the testis shows stage-dependent expression in the seminiferous epithelium (15). Similarly, the PTH2 receptor is also expressed in testis, pancreas, and placenta (16, 17). Here, in situ hybridization suggests a more complex pattern of expression with only light staining in the seminiferous epithelium and more pronounced staining in some interstitial cells and in sperm cells in the head of the epididymis. Surprisingly, a knockout of TIP39 apparently does not produce an obvious phenotype apart from sterility in both male and female Tifp39–/– progeny. The paper deals mainly with the testicular phenotype.
An undeniable merit of this paper is that it shows unambiguously that TIP39 is a novel regulatory molecule produced in the testis in vivo and essential to allow spermatocytes to complete meiosis. A hypothalamic site of action is made highly unlikely by the normal levels of LH and androgens and by the demonstration that selective restoration of TIP39 expression in germ cells by use of Cre/loxP technology overcomes the block in meiosis. The authors propose that TIP39 acts as an autocrine/paracrine agonist produced by the germ cells and activating PTH2 receptors also located on the germ cells. Not many regulatory molecules involved in germ cell-germ cell interactions have been identified, but this sounds like a reasonable hypothesis. Taking into account that both TIP39 expression and PTH2 expression increase dramatically between the time of the formation of the first spermatids (d 21) and adulthood, a regulatory system could be envisioned in which more mature germ cells support meiotic progression. It is obvious, however, that alternative interpretations merit consideration. The data do not show unambiguously the involvement of the PTH2 receptor in the control of meiosis. As already mentioned, this receptor is only lightly expressed in the seminiferous tubules and appears mainly on spermatozoa located in the head of the epididymis (16). Moreover, at the present stage of knowledge, expression of either TIP39 or PTH2 receptor by Sertoli cells cannot be ruled out completely. Moreover, as discussed by the authors, it remains intriguing that an apparently complete block in meiosis can be rescued by a system that restores expression of TIP39 under the ultimate control of the protamine promoter, a promoter that becomes active only at the spermatid stage and, accordingly, in cells that have successfully completed meiosis.
Another intriguing question raised by the mentioned study concerns the mechanism by which TIP39 may affect meiotic progression. In previously described examples of meiotic arrests, caused by defective expression of genes directly involved in chromosome recombination, DNA repair, or synapsis (Spo11, Dmc1, Msh4, Msh5, and Hop2), the state of alignment reached in a given nucleus is uniform (18, 19). This is not the case in Tifp39–/– mice in which about half of the chromosomes show homologous alignment and the other half do not. This may point to the fact that TIP39 is not directly or mechanistically involved in the process of meiosis but rather creates an environment that is permissive for meiotic progression. Although not investigated by the same techniques, the defect described here resembles the block in meiosis observed in mice with a Sertoli cell-selective knockout of the androgen receptor. Here too a nonhomogeneous arrest in meiosis is observed with fewer and fewer cells reaching more advanced stages of meiosis as reflected by a progressive reduction in the expression of the studied meiotic genes: Spo-11, Hsp70–2, SCP-3, and pro-acrosin-binding protein (8, 20).
The exact role of individual growth factors, hormones, and cytokines in the complex network of cell-to-cell communications in the testis remains poorly understood. From a technical point of view, the paper by Usdin et al. (11) points to the opportunities created by recent conditional knockout and expression systems including Cre/LoxP technology (21) to readdress several of the questions that have not come to a satisfactory response during the past decades. Systems allowing cell- or cell-lineage-selective and temporally controlled expression of local regulatory agents and their cognate receptors in the testis (or other tissues) are rapidly becoming available. Small promoter fragments of the anti-Müllerian hormone (22) and the reproductive homeobox on the X-chromosome 5 (23) genes have successfully been used to drive Sertoli cell-selective expression. Similarly, promoter fragments of the protamine (24), synaptonemal complex protein 1 (25), and tissue nonspecific alkaline phosphatase (26) genes have been used to specifically target male germ cells. The promoters tested to target selective expression in Leydig or peritubular myoid cells have not yet reached the desired degree of specificity. The use of bacterial artificial chromosome clones rather than short promoter fragments to drive gene expression may provide enhanced selectivity and better quantitative control (27). Variants of the Cre recombinase that become active only in the presence of an inducer such as tamoxifen increase the possibilities for temporal control (28). It may reasonably be expected that the availability of these tools will prompt renewed interest in the local control system in the testis and will ultimately help us to unravel the mechanisms underlying spermatogenic arrests and other disturbances in spermatogenesis presently classified as idiopathic.
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Abbreviation: TIP39, Tuberoinfundibular peptide of 39 residues.
Received May 20, 2008.
Accepted for publication May 21, 2008.
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