3.1. Morphology of Reproductive Tract Regions in Intact Prepubertal and Mature Hamsters
shows representative examples of control and neonatally DES-exposed complete tracts (vagina to ovaries) from prepubertal (d-21) and mature (2-mo) animals. At both ages, cervical and uterine horn dimensions were dramatically enhanced in DES-exposed animals. While gross appearance of the oviduct and ovary following neonatal DES treatment was not obviously affected in prepubertal animals, it was profoundly abnormal in mature animals due to severe inflammatory lesions of the oviduct and ovarian bursa (salpingitis).
Effects of neonatal DES treatment on complete reproductive tracts from prepubertal and mature hamsters
shows how the oviduct and ovary disruption phenomenon progresses at the histomorphological level. In prepubertal (day-21) animals, POF were more prevalent in the DES-exposed group but then less so in pubertal (1-month) animals. Thereafter, POF were rare in both groups. Beginning at puberty (see middle, right panel of ), cystic follicles were always found in DES-exposed animals. While vaginal estrous and multiple corpora lutea were observed in control animals at 2-months of age, neither were observed in DES-exposed animals at any age. Instead (see lower, right panel of ), a continued presence of cystic follicles and progressive disorganization of both follicle and stromal tissue compartments were observed in the ovaries of all neonatally DES-exposed animals.
Effects of neonatal DES treatment on the histomorphology of oviducts and ovaries in prepubertal and mature hamsters
also illustrates the severe inflammatory and tissue/organ-distorting impact of salpingitis that fulminates postpubertally in DES-exposed animals. Indeed, the extent of that condition at the gross level (as exhibited in ) often prevented us from finding the ovary in the massively inflamed tissue mass and/or it induced marked morbidity (abdominal distension, hunched/unstable gait, general muscular atrophy) and very early mortality in many of the DES-exposed animals.
3.2. Ovarian Transplantation Success
To test the alternative (direct vs. indirect) mechanisms of perinatal DES-induced ovarian disruption, we used the hamster cheek pouch site for ectopic organ transplantation. General performance of that site according to assessment of ovarian transplant mass viability at the gross inspection level (example in ) is presented in . Overall success rates ranged from 62% to 81% with no significant difference among the four transplant groups. Most of the successful transplant sites (~77%) were bilateral and, for those cases of unilateral success, there was no significant right vs. left bias with regard to neonatal treatment status of either the host or donor animals.
3.3. Vaginal Estrous Assessments
Hamster estrous cycles are monitored by a distinctive vaginal discharge (vaginal estrous) [41
] due to the action of ovarian progesterone secreted in response to the preovulatory LH surge. Strikingly, we observed vaginal estrous in all of the 16 control host animals (CON Homo or C/C and CON Cross or C/D groups) assessed but not in any of the 14 DES-exposed host animals (DES Homo or D/D and DES Cross or D/C groups) assessed, regardless of whether the cheek pouch transplants came from control or DES-exposed donors. Consistent with our unpublished but longstanding observations in DES-exposed intact animals, all of the DES-exposed host animals (DES Homo or D/D and DES Cross or D/C groups) also exhibited clear evidence of hypospadias [data not shown].
3.4. Endocrine Status
To assess general endocrine status in both control and neonatally DES-exposed groups of intact and surgically manipulated animals, we measured serum levels of estradiol (E2) () and luteinizing hormone (LH) plus follicle-stimulating hormone (FSH) (). In prepubertal animals (d-21), there was a trend of higher (2.0x) E2 levels and lower (0.7x) FSH levels while LH levels were significantly lower (0.1x) in the DES-exposed compared to the control group.
Effects of neonatal DES treatment on serum gonadotropin levels in prepubertal hamsters and in mature hamsters that were subjected or not to various prepubertal surgical procedures
In mature, intact animals (2-mo/I), serum E2 levels tended to be higher (3.0x) in the DES-exposed compared to the control group. Serum gonadotropin levels in such animals were generally low and not significantly different between the control and DES-exposed groups.
In mature, ovariectomized (2-mo/O) animals, serum E2 levels were barely detectable in both control and DES-exposed groups. Serum gonadotropin levels in such animals were: 1) generally elevated (LH/C - 12.3x; LH/D - 7.1x; FSH/C - 2.4x; FSH/D - 7.7x) compared to those in mature, intact (2-mo/I) animals, and 2) higher (LH - 1.8x; FSH - 2.6x) in DES-exposed compared to control animals, but that difference was significant only for the FSH assays.
In mature, ovariectomized, and estrogen-replaced (2-mo/O+E2) animals, E2 levels were: 1) equivalent in the control and DES-exposed groups, and 2) equivalent to those measured in the DES-exposed group of mature, intact animals (2-mo/I). In contrast, both LH (17.2x) and FSH (1.7x) levels were significantly higher in the DES-exposed group. This trend was similar to that noted for the 2-mo/O groups (but the enhanced LH levels in DES-exposed animals were more dramatic (9.6x) in the 2-mo/O+E2 group than in the 2-mo/O group).
In the mature host animals with viable ovarian masses in their cheek pouch transplantation sites, E2 levels in both the Homo- and Cross-transplant groups were: 1) almost identical in control vs. DES-exposed animals; and 2) equivalent to those in the control group of mature, intact animals (2-mo/I). Gonadotropin levels in both transplant groups were quite similar in control vs. DES-exposed animals and comparable between the two groups but differed in various ways from those measured in the other mature animal groups. For LH, levels in the transplant host animals were: 1) generally higher than those in both control (6.4x) and DES-exposed (2.1x) groups of mature, intact (2-mo/I) animals; 2) generally lower than those in the mature, ovariectomized (2-mo/O) animals and much more so in the DES-exposed (0.2x) than the control group (0.6x) of such animals; and 3) higher than in the control group (11.2x) but lower than in the DES-exposed group (0.5x) of mature, ovariectomized, and estrogen-replaced (2-mo/O+E2) animals. For FSH, levels in the transplant host animals were: 1) much higher than those in both control (6.1x) and DES-exposed (7.2x) groups of mature, intact (2-mo/I) animals; and 2) much higher than those in the control groups of both mature, ovariectomized (2-mo/O - 2.5x) and mature, ovariectomized, and estrogen-replaced (O+E2 − 2.4x) animals. Lastly, the similarity in serum E2, LH, and FSH levels between the control and DES-exposed groups of transplant host animals contrasts sharply with the differences observed between those groups in terms of vaginal estrous assessments presented above.
According to ANOVA evaluation among the mean values for all twelve treatment combinations shown in Figs. and : 1) E2 levels were significantly higher in all the 2-mo/O+E2 group animals than in all the animals in the 2-mo/O group and in both the Homo- and Cross-transplant groups; 2) LH levels were significantly higher in all the 2-mo/O group animals than in all the animals in the 2-mo/I group and in both the homo- and cross-transplant groups; and 3) FSH levels were significantly lower in all the 2-mo/I group animals than in all the animals in both the Homo- and Cross-transplant groups.
3.5. Uterine Dimensions and Histomorphology
To evaluate how and to what extent the normal and altered endocrine environments described above affected a terminal hormone target tissue, we assessed uterotrophic status, a well-established end-point of estrogenic responsiveness. Organ dimensions as determined here by uterine horn cross-sectional area measurements () were significantly enhanced (1.9 - 6.4x) in neonatally DES-exposed vs. control animals for all six treatment groups. In other words, regardless of whether circulating E2 levels were low (2-mo/O), high (2-mo/O+E2), or intermediate (Homo- and Cross-transplant hosts) but equivalent between the control and neonatally DES-exposed animals (), uterine dimensions were consistently enhanced in the DES-exposed animals. According to ANOVA evaluation among the mean values for all the twelve treatment combinations shown in , uterine dimensions were significantly less in all the d-21 and 2-mo/O group animals than in all the 2-mo/I and 2-mo/O+E2 group animals. We also noted (data not shown) that, in control and neonatally DES-exposed groups of mature (2-mo) host animals found not to have viable ovarian tissue transplants in either of their cheek pouches, their uteri mimicked the very atrophic state observed in control and neonatally DES-exposed groups of mature, ovariectomized (2-mo/O) animals, respectively, and is clearly indicative of estrogen withdrawal.
At the histopathological level, uterine aberrations in neonatally DES-exposed animals were strikingly similar among the mature, ovariectomized, and estrogen-replaced (2-mo/O+E2) group (, top panels) and both the Homo (, middle panels) and the Cross (, bottom panels) transplant host groups. Those aberrations were most dramatic in the endometrial epithelial compartment and consisted of extremely hyperplastic cells in a very chaotic pseudostratified organization that was riddled with cavities containing apoptotic cells (all three right-side panels in ). It is also noteworthy that the pattern of uterine hyperplasia/dysplasia that was manifest in a neonatal DES exposure-specific manner parallels that for the differences described above in terms of vaginal estrous assessments.
Effects of neonatal DES treatment on uterine histomorphology in mature, ovariectomized, and estrogen-replaced hamsters and in both groups of host hamsters with viable ovarian transplant tissue masses
3.6. In Situ and Transplant Ovary Histomorphology
Shown in are characteristic views of in situ ovaries (upper panels) and ectopic/transplanted ovaries (middle and lower panels) in mature (2-mo) hamsters. Complementing the evidence in , the upper panels in depict our consistent observations that: 1) multiple corpora lutea develop postpubertally within the in situ ovaries of control animals but never within those of neonatally DES-exposed animals and 2) cystic follicles are always found postpubertally within the in situ ovaries of DES-exposed animals. According to cross-section area analysis of the ovarian tissue regions present in the excised cheek pouch specimens (data not shown), mean dimensions of the transplant masses were not statistically different among the four treatment groups shown in the middle and lower panels of .
Histomorphological analysis of the viable transplant masses in all four treatment groups revealed the presence of ovarian tissue-containing follicles at various stages of development (); but none of the analyzed masses exhibited the normal ovarian histomorphology as depicted in the upper left panel of . In general, stromal compartments within the ovarian transplant masses were disorganized and most of the follicles appeared distorted. Furthermore, cells with the characteristic polyhedral morphology of healthy luteal cells were not observed in any of the transplant masses from the four treatment groups. However, structures reminiscent of the cystic follicles observed postpubertally within the in situ ovaries of DES-exposed animals (, upper right panel) were often observed within the transplant masses from both groups (5 of 11 D/D and 6 of 16 D/C) of neonatally DES-exposed hosts (, middle and lower right panels and , both right panels) but were seldom observed in both groups (1 of 11 C/C and 1 of 12 C/D) of control hosts (, middle and lower left panels and , both left panels). In other words, the ovarian disruption phenomenon of cystic follicle development at the cheek pouch transplant site depended on neonatal treatment history of the host animal rather than the donor animal.