Age-related changes in human testicular function

Main testicular functions are spermatogenesis and steroidogenesis. Androgen production, Testosterone (T) in particular, stimulates the differentiation of germ cells under the strict endocrine control of the pituitary gland via the two gonadotropins LH and FSH. Today we know that the reduction in serum T concentration (late onset hypogonadism) observed with age can be associated with an overall unhealthy status of the body. Moreover, the prevalence of obesity, metabolic syndrome and diabetes increases with age, and these conditions are significantly associated with hypogonadism, as assessed by sexual symptoms and low serum T levels in spite of normal LH. There is few information about mechanisms underlying androgen deficiency with age and little is known about human Leydig cell (LC) aging, in terms of number and function, mainly because of the scarce availability of the tissue. In this study, we evaluated the age-related changes in testicular function in healthy human testicular tissues of different ages (19-85 years) obtained from heart-beating organ donors and from patients referred to the andrology clinic having normal spermatogenesis and hormone levels. We demonstrated that aging is associated with changes in testicular morphology which are variable in different areas of the parenchyma. Changes included a significative reduction of the area occupied by seminiferous tubules coupled with an increased area occupied by interstitium, mainly composed by fibrotic tissue. In elderly donors, we noticed also peritubular thickening and the appearance of sclerotic tubules and empty tubules. Another morphological parameter evaluated was the LC micronodules frequency and size, since these aggregates composed by more than 15 LCs have been associated with a lower Testosterone/LH ratio. We did not find any correlation between age and micronodules distribution or size, pointing to a normal Hypothalamus-Pituitary-Gonad axis in our group of donors. We found a significative reduction of both LC and Sertoli cell (SC) number with age indicating that cellular senescence generally observed in many tissues with advancing age affects also the testis. Moreover, we evidenced a significative positive correlation between LC and SC number at all ages, which has not been described before in men. Concerning the function of LCs, we analysed gene expression of steroidogenic pathway enzymes and other LC markers by qRT-PCR. We did not find a significative correlation between enzyme gene expression and age but a negative trend was observed for 17β-HSD3, StAR and CYP11A1 genes. In contrast, INSL3 transcript, which also reflects LC number, significantly declined in aged men, consistent with our observation that LC population size changed during aging. Using the in vitro organ culture model previously developed in our laboratory, we demonstrated that three hours in vitro culture of both fresh and cryopreserved testis fragments allows to analyse the LC androgen production (testosterone (T), androstenedione (A), dehydroepiandrosterone (DHEA) and 17-Hydroxyprogesterone (17OHP)) secreted into the culture media and simultaneously measured by mass spectrometry both in basal conditions and under recombinant gonadotropin stimulation. We did not find any correlation between androgens secreted in basal conditions and age, indicating that LCs of the donors analysed were able to produce T, A, DHEA and 17OHP in an age- independent manner. The response to gonadotropin stimulation was found greatly variable among donors of different ages and not related to the age. The response to hCG was higher than that to LH when fresh tissue was used. Tissue cryopreservation did not alter interstitial compartment morphology but caused a significative reduction in T concentration, whereas A, DHEA and 17-OH-P levels increased, pointing to a particular vulnerability of specific enzymes to the freezing condition. These data are in line with the finding that the response to hCG was lost when the fragments were cultured after cryopreservation, reinforcing the idea that cryopreservation represents a stressor to the testicular tissue. In conclusion, our data point to a cellular senescence of the aging human testis which is not associated to the in vitro ability to produce androgens. Thus, our results support the idea that LC dysfunction is largely driven by aging of the whole testicular microenvironment rather than aging of LC population alone