DBP treatment during the masculinization programming window, but not right after it, causes break-up of seminiferous cords and formation of dysgenetic areas in rats

Reproductive disorders are extremely common nowadays and some studies show that their incidence may be increasing in the last decades. These disorders include cryptorchidism and hypospadias in newborns and low sperm counts, testis germ cell cancer and hypogonadism in young adult males. These disorders are hypothesized to comprise a Testicular Dysgenesis Syndrome (TDS) in which environmental and lifestyle factors are clearly implicated as potential causes. Although the TDS disorders manifest in different life stages (at birth or young adulthood), there is strong evidence showing that they may have a common origin in fetal life, pointing to the importance of mother’s lifestyle during pregnancy. As male reproductive development is a hormone dependent process, changes in the hormonal balance during fetal life, especially deficiency in androgen production and action during the masculinization programming window (MPW), can be related to these disorders. The MPW is thought to occur between 8 and 14 weeks in humans and from e15.5 to e18.5 in rats, a time period that is just after testis differentiation and seminiferous cord formation but before the differentiation of the reproductive tract which is highly dependent on androgens. Gestational exposure of pregnant rats to certain phthalate esters (such as dibutyl phthalate, DBP, which is a common environmental chemical) results in reproductive abnormalities in the male offspring that are very similar to TDS in humans, making this an excellent animal model for studying the fetal origins of human TDS. DBP treatment induces focal dysgenetic areas, which appear between e19.5-e21.5 and manifest as focal aggregation of Leydig cells and presence of ectopic Sertoli cells (SC), even when DBP treatment is initiated (e15.5) after completion of normal SC differentiation and seminiferous cord formation (e13.5-e14.5). There are some unexplained features about these ectopic SC: (1) they do not appear until beyond e19.5 (after cessation of DBP treatment); (2) DBP treatment during the period (e19.5-e21.5) when ectopic SC do appear, rather than during the MPW, fails to induce ectopic SC; (3) unlike normally differentiated SC, the ectopic SC do not form/initiate seminiferous cord formation during fetal life, but only later after birth. Our hypothesis is that the ectopic SC originate from breakdown of already formed seminiferous cords. To address this, time-mated female Wistar rats were treated daily with 750mg/kg/day of DBP in three different windows: full window (FW, e13.5-e20.5); masculinization programming window (MPW, e15.5-e18.5) or late window (LW, e19.5-e20.5). Fetal testis sections from the offspring were evaluated at several fetal ages, using triple immunofluorescence and stereology (n=6-12 per age/group). The results show that DBP treatment during the MPW produces more frequent and extensive dysgenetic areas, containing ectopic SC and germ cells (GC), while the LW treatment does not result in any dysgenetic areas. Additionally, the DBP treatment causes clustering of GC in the centre of the cords, especially in the FW and LW groups, while in the MPW group many GC still migrate to the basal lamina, as expected in normal testis development. The intensity of expression of smooth muscle actin, calponin and myosin in peritubular myoid cells at e21.5 in DBP exposed rats (MPW and FW) is reduced, suggesting an impaired basement membrane in these cords. Furthermore, we observed cords breaking up at e20.5 in the MPW group, releasing SC and GC to the interstitial compartment; we presume that this event gives rise to the focal dysgenetic areas observed in the FW and MPW DBP-treated testis. The mechanism for this still need to be investigated, and it might give us better understanding about how dysgenesis arises in human cases of TDS. Also, we show for the first time that a disruption can be induced after the testis is completely formed, and reinforced the critical importance of the MPW in this process.