Transgenerational effects of environmental toxins require either a chromosomal or epigenetic alteration in the germ line. Transient exposure of a gestating female rat during the period of gonadal sex determination to the endocrine disruptors vinclozolin (an antiandrogenic compound) or methoxychlor (an estrogenic compound) induced an adult phenotype in the F 1 generation of decreased spermatogenic capacity (cell number and viability) and increased incidence of male infertility. These effects were transferred through the male germ line to nearly all males of all subsequent generations examined (that is, F 1 to F 4 ). The effects on reproduction correlate with altered DNA methylation patterns in the germ line. The ability of an environmental factor (for example, endocrine disruptor) to reprogram the germ line and to promote a transgenerational disease state has significant implications for evolutionary biology and disease etiology.

Morphological male sex determination is dependent on migration of endothelial and preperitubular cells from the adjacent mesonephros into the developing testis. Our hypothesis is that VEGFA and its receptor KDR are necessary for both testicular cord formation and neovascularization. The Vegfa gene has 8 exons with many splice variants. Vegfa120, Vegfa164, and Vegfa188 mRNA isoforms were detected on Embryonic Day (E) 13.5 (plug date = E0) in the rat. Vegfa120, Vegfa144, Vegfa164, Vegfa188, and Vegfa205 mRNA were detected at E18 and Postnatal Day 3 (P3). Kdr mRNA was present on E13.5, whereas Fms-like tyrosine kinase 1 receptor (Flt1) mRNA was not detected until E18. VEGFA protein was localized to Sertoli cells at cord formation and KDR to germ and interstitial cells. The VEGFA signaling inhibitors SU1498 (40 μM) and VEGFR-TKI (8 μM) inhibited cord formation in E13 testis cultures with 90% reduced vascular density (P < 0.01) in VEGFR-TKI-treated organs. Furthermore, Je-11 (10 μM), an antagonist to VEGFA, also perturbed cord formation and inhibited vascular density by more than 50% (P < 0.01). To determine signal transduction pathways involved in VEGFA's regulation of testis morphogenesis, E13 testis were treated with LY 294002 (15 μM), a phosphoinositide 3-kinase (PI3K) pathway inhibitor, resulting in inhibition of both vascular density (46%) and cord formation. Thus, we support our hypothesis and conclude that VEGFA, secreted by the Sertoli cell, is involved in both neovascularization and cord formation and potentially acts through the PI3K pathway during testis morphogenesis to elicit its effects.

Depletion of the ovarian reserve is associated with reproductive senescence in mammalian females, and there is a positive relationship between the size of the ovarian reserve and the number of antral follicles on the surface of the ovary. Therefore, we conducted a series of experiments to investigate the influence of stage of the estrous cycle, age, and birth weight on antral follicle counts (AFC) in beef cows and heifers. Pairs of ovaries were collected from crossbred beef cows at slaughter (n = 72) or at necropsy (n = 333; 0 to 11 yr of age); all visible antral follicles were counted, the ovaries were weighed, and stage of the estrous cycle was estimated based on ovarian morphology. There was no influence of estimated stage of the estrous cycle on AFC (P = 0.36). There was a small but positive effect of birth weight on AFC [AFC = -1.7 + 0.31(birth weight); P = 0.007, r(2) = 0.05]. When antral follicle counts were regressed on age, there was a quadratic effect of age such that AFC increased until 5 yr of age and decreased thereafter [AFC = 12.9 + 9.0(yr) - 0.86(yr(2)); P < 0.001, r(2) = 0.22]. In a third experiment, crossbred beef heifers (n = 406; 353 to 463 d of age) at 3 locations were subjected to ovarian ultrasonography on unknown day of the estrous cycle. Heifers were classified as low AFC (<15 follicle, n = 84) or high AFC (>24 follicles, n = 178). Whereas estimated stage of the estrous cycle did not influence AFC (P = 0.62), heifers classified as low AFC had smaller ovaries (P = 0.001), decreased birth weight (P = 0.003), and a decreased heifer pregnancy rate (P = 0.05) compared with heifers in the high AFC group. From these results, we conclude that AFC in beef cows and heifers is influenced by birth weight and age but not by stage of the estrous cycle. In beef cows, the number of antral follicles increases to 5 yr of age and then begins to decline. This may indicate that a decrease in fertility due to decline of the ovarian reserve may begin earlier than previously thought in beef cows.

The process of seminiferous cord formation is the first morphological event that differentiates a testis from an ovary and indicates male sex determination. Cord formation occurs by embryonic Day 14 (Day 0 = plug date; E14) in the rat. A series of experiments were conducted to determine if neurotropins and their receptors are important for the process of rat embryonic cord formation. The expression of low affinity neurotropin receptor (p75/LNGFR) was determined by immunohistochemistry on sections of both testis and ovary from E13 through birth (Day 0, P0) with an antibody to p75/LNGFR. The staining for p75/LNGFR was present in the mesonephros of E13 gonads and in a sex-specific manner appeared around developing cords at E14 in the embryonic testis. At birth, staining for p75/LNGFR was localized to a single layer of cells (i.e., peritubular cells) that surrounded the seminiferous cords. The genes for both neurotropin 3 (NT3) and for corresponding high affinity neurotropin trkC receptor were found to be expressed in the E14 rat testis, as well as other neurotropins and receptors. Immunocytochemical analysis of E14 rat testis demonstrated that NT3 was localized to the Sertoli cells and trkC was present in individual cells of the interstitium at E16 and in selected preperitubular cells at E18. Previously, the peritubular cells adjacent to the cords were demonstrated to be derived from migrating mesonephros cells around the time of cord formation. To determine if neurotropins were involved in cord formation, the actions of neurotropins were inhibited. A high affinity neurotropin receptor (trk)-specific kinase inhibitor, K252a, was used to treat organ cultures of testes from E13 rats prior to cord formation. Treatment of E13 testis organ cultures with K252a completely inhibited cord formation. K252a-treated organ cultures of E14 testis that contained cords did not alter cord morphology. A second experiment to inhibit neurotropin actions utilized a specific antagonist trk-IgG chimeric fusion protein and E13 testis organ cultures. The trk-IgG molecules dimerize with endogenous trk receptors and inhibit receptor signaling and activation of ligand function. Forty percent of E13 testis organ cultures treated with trkC-IgG had significantly reduced cord formation. TrkA-IgG had no effect on initiation of cords; however, in fifty percent of the treated organs, a "swollen" appearance of the cord structures was observed. Experiments using trkB-IgG chimeric protein on E13 organ cultures had no effect on cord formation or cord morphology. The testes from trkC and NT3 knockout mice were examined to determine if there were any morphological differences in the testis. NT3 knockouts appeared to have normal cord morphology in E15 and E17 testis. TrkC knockout mice also had normal cord morphology in E14 and P0 testis. Both NT3 and trkC knockout-mice testis had less interstitial area than wild-type controls. In addition, the trkC knockout mice have an increased number of cells expressing p75LNGFR within the cords when compared to control...

We hypothesized that vascular endothelial growth factor A (VEGFA) angiogenic isoforms and their receptors, FLT1 and KDR, regulate follicular progression in the perinatal rat ovary. Each VEGFA angiogenic isoform has unique functions (based on its exons) that affect diffusibility, cell migration, branching, and development of large vessels. The Vegfa angiogenic isoforms (Vegfa_120, Vegfa_164, and Vegfa_188) were detected in developing rat ovaries, and quantitative RT-PCR determined that Vegfa_120 and Vegfa_164 mRNA was more abundant after birth, while Vegfa_188 mRNA was highest at Embryonic Day 16. VEGFA and its receptors were localized to pregranulosa and granulosa cells of all follicle stages and to theca cells of advanced-stage follicles. To determine the role of VEGFA in developing ovaries, Postnatal Day 3/4 rat ovaries were cultured with 8 muM VEGFR-TKI, a tyrosine kinase inhibitor that blocks FLT1 and KDR. Ovaries treated with VEGFR-TKI had vascular development reduced by 94% (P < 0.0001), with more primordial follicles (stage 0), fewer early primary, transitional, and secondary follicles (stages 1, 3, and 4, respectively), and greater total follicle numbers compared with control ovaries (P < 0.005). V1, an inhibitor specific for KDR, was utilized to determine the effects of only KDR inhibition. Treatment with 30 muM V1 had no effect on vascular density; however, treated ovaries had fewer early primary, transitional, and secondary follicles and more primary follicles (stage 2) compared with control ovaries (P < 0.05). We conclude that VEGFA may be involved in primordial follicle activation and in follicle maturation and survival, which are regulated through vascular-dependent and vascular-independent mechanisms.