Population Council Research that makes a difference

Population Council studies focus on those growth factors mediating intracellular communication between Sertoli cells and developing germ cells, messages that are essential for the coordinated regulation of sperm production. Within the seminiferous tubule, Sertoli cells act in concert with specific sets of germ cells. Sertoli cells produce interferons, interleukins, and several stem cell growth factors. These products target specific receptors on the surfaces of both germ cells (paracrine regulation) and the Sertoli cell itself (autocrine signaling).

Recent Council studies implicate the immediate early genes or proto-oncogenes as initial responses to growth factors within the seminiferous tubule. Characterization of the cellular binding sites and the distinct transcription factors that mediate the effects of these growth factors is anticipated to lead to the design of new, therapeutic male fertility drugs. It is also anticipated that elucidation of these pathways in both particular germ cells and in Sertoli cells will lead eventually to the design of new compounds to interfere with sperm production and function as safe, reversible male contraceptives.

Leukemia inhibitory factor (LIF) is a growth factor shown to enhance the survival of rapidly proliferating cells in the hematopoietic system, human breast cancer cells, mouse embryonic stem cells, and more recently, rat testicular gonocytes and Sertoli cells. Research efforts have been focused on defining the testicular source(s) and specific cellular targets for LIF. The membrane-binding complex that specifically recognizes LIF is composed of two chains, LIF receptor (LIFR) and gp130. In 1999, Council researchers demonstrated the expression of LIF, LIFR, and gp130 mRNAs in both Sertoli cells and specific germ cells (spermatogonia, pachytene, round, and elongated spermatids), data suggestive of multiple cell-specific LIF-mediated functions. Researchers characterized the effects of LIF on the signal transducers and activators of transcription (STAT)-3 and STAT-1, oncogene expression, and activator protein-1 (AP-1) regulation in Sertoli cells.

Council scientists next determined that follicle-stimulating hormone (FSH), cyclic AMP (cAMP), and LIF elicit substantial changes in the metabolism of retinol (vitamin A) in Sertoli cells. It was shown that the addition of cAMP or FSH to primary cultures of purified rat Sertoli cells results in a two- to three-fold increase in the metabolism of retinol to retinoic acid; the metabolism of retinol to retinyl esters, especially retinyl palmitate, was also increased by approximately five-fold relative to untreated Sertoli cells. In contrast, LIF addition results in a reduction of the rate of retinol metabolism, while levels of retinyl palmitate increased relative to levels in untreated Sertoli cells. Culture of the Sertoli cells in the presence of all-trans retinoic acid also increased the rate of retinol metabolism.

However, in this case the metabolism of retinol to retinoic acid was inhibited, while the metabolism of retinol to retinyl esters was increased by over 50-fold. Concomitant with the increase in retinoic acid synthesis from retinol induced by cAMP or FSH, a dramatic increase (>40-fold) in aldehyde dehydrogenase-2 (AHD-2) mRNA was observed. The AHD-2 enzyme can metabolize retinaldehyde to retinoic acid. Very low levels of retinaldehyde dehydrogenase-2 (RALDH-2) mRNA and the cytochrome P450 CYP26 retinoic acid hydroxylase [CYP26(RAH)] mRNA were seen in the Sertoli cells under all treatment conditions tested. Cellular retinoic acid-binding protein-I and -II (CRAB-I, -II, respectively) and the retinoic acid receptor RAR-beta mRNA levels were also very low.

These results indicate that one function of FSH (and/or cAMP) in Sertoli cells is to increase the metabolism of retinol to the biologically active metabolite retinoic acid and to retinyl esters. Moreover, the increase in AHD-2 mRNA following FSH demonstrates that AHD-2 is a biomarker for FSH action in Sertoli cells. Studies are now focused on the differential transcriptional activation of the retinoid receptors by particular IL6 cytokines in Sertoli cells and early germline and stem cells.

Thus, these studies demonstrated that:

• LIF is expressed within the seminiferous tubule of the pubertal and adult testis;
• the testicular LIF signaling pathway is contingent upon the phosphorylation of latent transcription factors; and
• specific gene expression and retinol metabolism are regulated by LIF. These data are consistent with unique LIF-mediated signaling events involving both somatic and germ cells during spermatogenesis