Depo-Provera increases neural activity in the central amygdala of reindeer bulls.

Reindeer bulls are difficult to manage and dangerous to handlers during the rutting period. Progesterone agonists have been used anecdotally in the field to favorably influence behavior, but effects on reproductive signaling have not been determined. The objective of this study was to determine the effects of Depo-Provera (medroxyprogesterone acetate) on neural activity in the amygdala of reindeer bulls in the early (n = 4) and full (n = 4) rut. Treated bulls (n = 4) were injected with a single injection of Depo-Provera (400 mg i.m.) approximately 2 wk before rut was initiated. Control bulls were untreated. Bulls were exsanguinated and brains collected. Neural activity in the amygdala was determined using c-fos immunohistochemistry. Neural activity did not differ by treatment (P ≥ 0.5), collection period (P ≥ 0.5), or their interaction (P ≥ 0.3) in the medial and cortical amygdala nuclei. A treatment × time interaction (P = 0.009) was observed in the central amygdala. The amygdala nuclei are interconnected allowing for integration of sensory stimuli with a direct connection between the medial amygdala and the olfactory bulb. The central amygdala is responsible for alerting, fear, and initiating a state of arousal towards nonspecific stimuli in the surrounding environment. In wildlife, the central amygdala has a role in recognizing threats in the environment such as predators. During the rut, bulls normally have a decreased sense of fear and elevated aggressive behavior with Depo-Provera treatment seemly able to diminish that aggression. Although it is unlikely that this observed change in neural activity fully explains the decreased aggressive behavior noted in bulls treated with Depo-Provera, neural networks of aggression include the amygdala. It is possible that further changes in c-fos activity will be noted in other areas of the brain known to be necessary for processing social cues. Bulls treated with Depo-Provera maintain sexual interest and have offspring. Depo-Prevera increases the neural activity within the central amygdala and may partially account for their altered aggressive behavior during the rut.

A polymorphism in myostatin influences puberty but not fertility in beef heifers, whereas µ-calpain affects first calf birth weight.

The use of genetic markers to aid in selection decisions to improve carcass and growth characteristics is of great interest to the beef industry. However, it is important to examine potential antagonistic interactions with fertility in cows before widespread application of marker-assisted selection. The objective of the current experiment was to examine the influence of 2 commercially available markers currently in use for improving carcass traits, the myostatin (MSTN) F94L and µ-calpain (CAPN1) 316 and 4751 polymorphisms, on heifer development and reproductive performance. In Exp. 1, beef heifers (n = 146) were evaluated for growth and reproductive traits over a 3-yr period to determine if these polymorphisms influenced reproductive performance. In Exp. 2, heifers representing the 2 homozygous genotypes for the MSTN F94L polymorphism were slaughtered on d 4 of the estrous cycle and reproductive tracts were collected for morphological examination. In Exp. 1, there was a tendency (P = 0.06) for birth BW to be affected by MSTN with the Leu allele increasing birth BW in an additive fashion. Additionally, MSTN significantly affected the proportion of pubertal heifers by the start of the breeding season (P < 0.05) with the Leu allele additively decreasing the proportion pubertal; however, this did not result in a delay in conception or a decrease in pregnancy rates during the first breeding season (P > 0.15). The GT haplotype of CAPN1, which was previously associated with decreased meat tenderness, was associated with an additive decrease in birth BW of the first calf born to these heifers (P < 0.05). In Exp. 2, there were no differences between the MSTN genotypes for gross or histological morphology of the anterior pituitary, uterus, or ovaries (P > 0.05). From these results, we concluded that the MSTN F94L and CAPN1 polymorphisms can be used to improve carcass traits without compromising fertility in beef heifers. The influence of these markers on cow performance and herd life remains to be determined. While the delay in puberty associated with the MSTN F94L polymorphism did not negatively impact reproductive performance in heifers, caution should be used when combining this marker with other markers for growth or carcass traits until the potential interactions are more clearly understood.

Heifer development systems: a comparison of grazing winter range or corn residue.

Two experiments at 2 Nebraska locations evaluated effects of heifer development system on growth and pregnancy rate. In Exp. 1, heifers (n=270, BW=225 ± 2 kg) grazed winter Sandhills range (WR) or west central Nebraska corn residue (CR) with a supplement (0.45 kg/animal; 31% CP; 80 mg·animal(-1)·d(-1) of monensin). In Exp. 2, heifers (n=180, BW = 262 ± 3 kg) grazed eastern Nebraska WR or CR with a supplement (0.45 to 0.90 kg/d; 31% CP; 80 to 160 mg·animal(-1)·d(-1) of monensin). The CR heifers tended to have less (P=0.10) ADG compared with WR heifers before breeding in Exp. 1; however, prebreeding ADG was similar (P=0.77) in Exp. 2. Prebreeding BW, percentage of mature BW at breeding, and pregnancy determination BW were similar (P ≥ 0.14) for CR and WR in both experiments. Percentage of heifers pubertal at breeding, AI conception, and AI pregnancy rate (Exp. 2) and final pregnancy rate in both experiments were also similar (P ≥ 0.27) for CR and WR heifers. Precalving BW, percentage of calves born in the first 21 d, calf birth date, calf birth BW, and dystocia score were all similar (P ≥ 0.21) for CR and WR heifers in both experiments. Cow BW at weaning, calf weaning BW, adjusted 205-d calf BW, and second season pregnancy rates were not affected (P ≥ 0.16) by treatment. Heifer development system did not affect (P ≥ 0.56) the cost of producing 1 pregnant heifer in Exp. 1 or 2. Development on CR may reduce ADG before breeding, but did not affect pregnancy rate. Heifer development using CR or WR postweaning resulted in similar reproductive performance and development cost.

Effect of dietary crude protein source on hormone and follicle characteristics in beef heifers.

Ground, raw soybeans (SB), or dried distillers grain plus solubles (DDGS) were utilized in heifer development diets to determine the effect of dietary fat and protein source on hormone and follicle characteristics and ADG. The experiment was conducted over 2 yr with 100 June-born heifers (199 +/- 2 kg initial BW, n = 50 per yr). The experimental periods were 157 and 207 d in yr 1 and 2, respectively. Heifers were provided a dietary supplement (DM basis) of 1.23 kg of SB and 0.40 kg of corn or 1.65 kg of DDGS between weaning and breeding. Estrus was synchronized with 2 injections of PGF(2alpha) 14 d apart. Dominant follicles were measured and aspirated via transvaginal ultrasonography 60 h after the second PGF(2alpha) injection. Heifers were exposed to bulls beginning 14 d after aspiration for 45 d. Heifer ADG was greater (P = 0.02) for DDGS heifers in yr 1, but was similar (P = 0.47) in yr 2. However, there was no difference (P = 0.35) in final BW in either year. There was no difference (P >or= 0.67) in follicle size, follicle hormone concentrations, or pregnancy rate (88%) between yr 1 and 2. Serum estrogen at 48 or 60 h after PGF(2alpha) injection were similar (P >or= 0.91); however, LH at 60 h in yr 2 tended to be greater (P = 0.07) for DDGS heifers. The percentage of heifers experiencing an LH surge 48 and 60 h after PGF(2alpha) injection was not affected (P >/= 0.40) by treatment. Calf production was not affected (P >or= 0.20) by developmental diet. In summary, DDGS and SB have similar effects on hormone and follicle characteristics at the inclusion rates used in these studies.

Evaluation of antral follicle count and ovarian morphology in crossbred beef cows: investigation of influence of stage of the estrous cycle, age, and birth weight.

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.

A proposed role for VEGF isoforms in sex-specific vasculature development in the gonad.

Many scientists have expended efforts to determine what regulates development of an indifferent gonad into either a testis or ovary. Expression of Sry and upregulation of Sox9 are factors that initiate formation of the testis-specific pathway to allow for both sex-specific vasculature and seminiferous cord formation. Migration of mesonephric precursors of peritubular myoid cells and endothelial cells into the differentiating testis is a critical step in formation of both of these structures. Furthermore, these events appear to be initiated downstream from Sry expression. Sertoli cell secretion of growth factors acts to attract these mesonephric cells. One hypothesis is that a growth factor specific for these cell linages act in concert to coordinate migration of both peritubular and endothelial cells. A second hypothesis is that several growth factors stimulate migration and differentiation of mesonephric 'stem-like' cells to result in migration and differentiation into several different cell lineages. While the specific mechanism is unclear, several growth factors have been implicated in the initiation of mesonephric cell migration. This review will focus on the proposed mechanisms of a growth factor, Vascular Endothelial Growth Factor, and how different angiogenic and inhibitory isoforms from this single gene may aid in development of testis-specific vascular development.

Utilization of soybeans or corn milling by-products in beef heifer development diets.

Whole raw soybeans (SB), wet corn gluten feed (WCGF), and corn dried distillers grains (DDG) are sources of protein in heifer development rations. The objectives of this study were to compare puberty status before synchronization of estrus, response to synchronization, and AI and final pregnancy rates in heifers developed on diets containing SB, WCGF, or DDG that were formulated to be similar in energy and CP. These ingredients vary substantially in fat content, which may affect reproductive performance. Rate of gain during the feeding period and post-AI performance were also compared. In a preliminary experiment, 104 crossbred heifers were fed diets containing either 1.25 kg of SB/d or 2.0 kg of WCGF/d for 110 d (DM basis), beginning at 10 mo of age. In Exp. 1, 100 crossbred heifers received either 1.25 kg of SB/d or 2.5 kg of WCGF/d from approximately 7 to 10 mo of age (91 d; 4 pens/diet), and then were fed 1.25 kg of SB/d for an additional 114 d (4 pens/diet). In Exp. 2, 1.25 kg of SB/d or 1.25 kg of DDG/d was fed to 100 crossbred heifers for 226 d, beginning at 6 mo of age (4 pens/diet). At approximately 13 mo of age, heifers were fed melengestrol acetate (0.5 mg/d) for 14 d, followed by an i.m. injection of PGF(2 alpha) (25 mg) 19 d later to synchronize estrus. Heifers (14 mo of age) received AI for 5 d after PGF(2 alpha), at which time the dietary treatments were ended. Heifers were commingled while grazing on native pasture and were exposed to bulls for approximately 60 d beginning 10 d after the last day of AI. Pregnancy to AI was determined by ultrasound 45 d after the last day of AI. Heifers fed SB in the preliminary experiment had a lower (P < 0.05) synchronization rate (81 vs. 96%) and longer interval (P = 0.05) from PGF(2 alpha) to estrus (76.6 vs. 69.2 h) compared with heifers fed WCGF. In Exp. 1, the age at which the heifers were begun on SB diets did not alter (P > 0.10) the synchronization rate (79%) or timing of estrus after PGF(2 alpha) (77.8 h). In Exp. 2, the synchronization rate (86%) and timing of estrus after PGF(2 alpha) (69.3 h) did not differ (P > 0.10) because of diet. No differences (P > 0.10) were due to diet for AI conception rates (overall mean for each experiment: 76.5, 60, and 68.5%), percentage of all heifers becoming pregnant to AI (67, 46, and 59%), or final pregnancy rates (92, 90, and 90%) in the preliminary experiment, Exp. 1, or Exp. 2, respectively. In summary, SB, DDG, and WCGF can be used as sources of protein in heifer development diets at the inclusion rates used in these studies.

Utilization of dried distillers grains for developing beef heifers.

A 2-yr study was conducted at 2 locations to determine if supplementing beef heifers with dried distillers grains (DDG) as an energy source affected growth or reproduction. Spring-born crossbred heifers (n = 316) were blocked by age or sire and age and assigned randomly to DDG or control (dried corn gluten feed, whole corn germ, urea) supplement. Heifers received prairie hay in amounts sufficient for ad libitum intake and 0.59% of BW DDG or 0.78% of BW control supplement (DM basis). Supplements were formulated to be isocaloric, but protein degradability differed. Supplemental undegradable intake protein intake from DDG averaged 267 g/animal daily and reached 318 g/animal daily; control supplemental undegradable intake protein intake averaged 90 g/animal daily and peaked at 107 g/animal daily. Initial pubertal status was determined by 2 blood samples collected 10 d apart, and monthly BW were collected from November through January; then biweekly BW and blood samples were collected from February until May yearly. Heifers were synchronized with 2 injections of PGF2alpha 14 d apart; estrus was detected and heifers were artificially inseminated for 5 d and placed with bulls 10 d later. Conception and pregnancy rates were determined via transrectal ultrasonography. Initial age, BW, and BCS did not differ (P > 0.92) for control and DDG heifers. Final BW, ADG, and final BCS also were not affected (P > 0.31) by supplementation. Estimated age and BW at puberty did not differ (P > 0.23) between treatments, and the proportions of pubertal heifers did not differ at the initiation of the experiment (P > 0.82), at the beginning of the 14-d sampling intervals, or before synchronization. Estrus synchronization rate (75.9%), time of estrus, and overall pregnancy rate (89.5%) were not affected (P > 0.14) by treatment. However, a greater proportion (P = 0.008) of DDG than control heifers conceived to AI (75.0 vs. 52.9%), resulting in greater (P = 0.07) AI pregnancy rates for DDG heifers (57.0 vs. 40.1%). Body weight or BCS at pregnancy diagnosis did not differ (P > 0.52) between DDG and control heifers. Supplementing beef heifers with DDG during development did not affect age at puberty but improved AI conception and pregnancy rates compared with an isocaloric control supplement.

Frequency of luteinizing hormone pulses in cattle influences duration of persistence of dominant ovarian follicles, follicular fluid concentrations of steroids, and activity of insulin-like growth factor binding proteins.

The objectives of the present study were to determine how varying frequency of LH pulses as controlled by varying treatments with progesterone (P4) in cattle would affect: (1) concentration of steroid hormones and activity of insulin-like growth factor binding proteins (IGFBPs) in the ovarian follicular fluid and blood plasma, and (2) duration of persistence of largest ovarian follicles. There were four treatment groups (n=7 per group) and a control group (n=5) of mature, non-lactating beef cows. Treatments were: (1) two progesterone releasing intravaginal devices (PRIDs) for 16 days (2PRID); (2) a half PRID for 16 days (0.5PRID); (3) two PRIDs for 8 days, then a half PRID for 8 days (2-0.5PRID); or (4) a half PRID for 8 days, then two PRIDs for 8 days (0.5-2PRID). Treatment was initiated on the fifth day of the estrous cycle, which was designated as Day 0, and continued for 16 days. All P4-treated females were administered prostaglandin F2alpha on Day 0 and 1 to regress their corpora lutea. Frequency of LH pulses was greater during treatment with the smaller dose of P4 compared with treatment with the larger dose of P4 and the control group. Ovarian follicles were classified into five categories based on ultrasonographic observations: growing (G); atretic (A); growing dominant (GD); growing persistent (GP); or atretic persistent (AP). At ovariectomy on Day 16, the largest and second largest follicles collected were re-classified into five categories based on follicular concentration of steroids. Classification of the largest follicle collected on Day 16 was influenced by treatment (P<0.005), with the 2PRID group having A follicles, the 2-0.5PRID group GP follicles, the 0.5-2PRID group AP follicles, and the 0.5PRID group GD and GP follicles. Concentrations of 17beta-estradiol (E2) were greatest in GD and GP follicles (P<0.05). There was less (P<0.05) activity of IGFBP-2 in GD follicles and less (P<0.05) activity of IGFBP-3 in GD and GP follicles than other follicles. Activity of IGFBP-4 and -5 was greater (P<0.05) in A and AP follicles than G, GD, and GP follicles. Maintenance of a frequent release of LH pulses over a 16-day period did not result in maintenance of persistent follicles throughout this period indicating that duration of dominance of these follicles is finite even when there is frequent release of LH pulses. Follicular atresia is associated with greater activity of IGFBP-2, -4, -5, and greater concentrations of P4 in follicles, whereas growing dominant and persistent follicles contained greater concentrations of E2, androstenedione (A4), and less IGFBP-2 activity than follicles of other classes. Follicle classifications based on ultrasonography or follicular concentration of steroids did differ (P<0.05) for the largest follicles from the 2PRID group. Two follicles in this group appeared as GD follicles by ultrasonography, but these were atretic based on follicular steroid contents. Objective 1 of the present study yielded the conclusion that concentrations of steroid hormones in follicular fluid and blood plasma could be predictably controlled by regulating the frequency of LH pulses with varying doses of P4. Objective 2 yielded the conclusion that maintain frequent release of LH pulses over a 16-day period could not maintain persistent follicles throughout this period, indicating that duration of dominance of these follicles is finite even when there is frequent release of LH pulses. Follicular atresia in the present study was associated with increased follicular fluid activity of IGFBP-2, -4, -5, and P4, whereas growing dominant and persistent follicles contained greater concentrations of E2, A4, and less IGFBP-2 activity than follicles of other classes.

Expression, action, and regulation of transforming growth factor alpha and epidermal growth factor receptor during embryonic and perinatal rat testis development.

The objective of the current study was to extend previous observations and examine the expression pattern and effects of transforming growth factor alpha (TGFalpha) and epidermal growth factor receptor (EGFR) on embryonic testis morphogenesis and growth. The expression of TGFalpha was determined after morphological sex determination (seminiferous cord formation at embryonic day 13 [ED13]) through perinatal testis development (postnatal day 5 [PD5]) with a quantitative reverse transcription-polymerase chain reaction procedure. Expression of messenger RNA (mRNA) for TGFalpha appeared to be more dynamic during testis development when compared with the expression of mRNA for EGFR. Message for TGFalpha was reduced at ED16 and PD4, and was elevated at PD0 during testis development. In contrast, EGFR mRNA levels were negligible at ED15 and were elevated constitutively from ED16 through PD5. Immunohistochemistry was conducted at ED14, ED16, ED19, PD0, PD3, and PD5 to localize cellular expression of both TGFalpha and EGFR. At ED16, positive staining for EGFR was localized to the cords, and by ED19, was mainly in the cords with slight expression in the interstitium. From PD0 to PD5, positive staining for EGFR was detected in the germ, Sertoli, and interstitial cells. Immunohistochemistry for TGFalpha detected localization at ED14 and ED16 to the Sertoli cells and to specific cells in the interstitium. From ED19 through PD5, TGFalpha was detected in the Sertoli, germ, and interstitial cells, and in endothelial cells within the interstitium. To determine the effects of TGFalpha on embryonic testis growth and seminiferous cord formation, ED13 testis organ cultures were treated with sense and antisense TGFalpha oligonucleotides. Antisense TGFalpha inhibited testis growth by 25%-30% in ED13 testis organ cultures when compared with sense oligonucleotide control pairs. To examine the effects of TGFalpha on perinatal testis growth, PD0 testis cultures were treated with different doses of TGFalpha. TGFalpha increased thymidine incorporation into DNA in PD0 testis cultures. Therefore, TGFalpha appears to have actions on both embryonic and perinatal testis growth. The regulation of TGFalpha and EGFR mRNA levels were examined using PD0 testis cultures treated with hormones that stimulate testis growth. Follicle-stimulating hormone (FSH) stimulated (P < .05) and testosterone tended to stimulate (P < .07) mRNA expression of EGFR. Epidermal growth factor stimulation of PD0 testis cultures did not affect levels of mRNA expression for EGFR, but did suppress expression of mRNA for TGFalpha. These results taken together demonstrate that TGFalpha can act to regulate early embryonic and perinatal testis growth. Furthermore, TGFalpha and EGFR expression can be regulated through growth stimulatory hormones such as FSH and testosterone.

Actions of the endocrine disruptor methoxychlor and its estrogenic metabolite on in vitro embryonic rat seminiferous cord formation and perinatal testis growth.

The current study examines the actions of methoxychlor and its estrogenic metabolite, 2, 2-bis-(p-hydroxyphenyl)-1, 1, 1-trichloroethane (HPTE), on seminiferous cord formation and growth of the developing rat testis. The developing testis in the embryonic and early postnatal period is likely more sensitive to hormonally active agents than at later stages of development. Embryonic day 13 (E13) testis organ cultures were treated with either 0.2, 2, or 20 microM methoxychlor or 1, 3, 6, 15, 30, or 60 microM HPTE to examine effects on cord formation. No concentration of methoxychlor completely inhibited cord formation. However, cord formation was abnormal with the presence of a reduced number of cords and appearance of "swollen" cords at the 2 and 20 microM concentrations of methoxychlor. The swollen cords were due to an increase in the number of cells in a cord cross section and reduction of interstitial cell numbers between cords. Treatment of embryonic day 13 (E13) testes with HPTE caused abnormal cord formation at the 3 microM and 6 microM concentrations, and completely inhibited cord formation at the 15, 30, and 60 microM concentrations. In addition to the estrogenic metabolite HTPE, methoxychlor can also be metabolized into anti-androgenic compounds. Therefore, to determine the spectrum of potential actions of methoxychlor on testis development, different concentrations of estradiol, testosterone, and an anti-androgen (flutamide) were utilized to determine their effects on E13 testis organ culture morphology. Estradiol (1 microM) and flutamide (0.1microM) both inhibited seminiferous cord formation in E13 testis organ cultures. Therefore, methoxychlor may be acting through the androgen and/or estrogen receptors to elicit its actions on seminiferous cord formation. Reverse transcription polymerase chain reaction (PCR) (RT-PCR) confirmed the presence of estrogen receptor alpha (ERalpha) mRNA from embryonic day 14 (E14) through postnatal day 5 (P5) while estrogen receptor beta (ERbeta) mRNA did not appear until approximately E16 of testis development. Androgen receptor (AR) expression was present from E14 through P5 of testis development, but at apparently reduced levels at E14 and E16. Immunohistochemical analysis localized ERalpha to the cells of the seminiferous cords at E14 though P5 while ERbeta was present in cells of the interstitium at E16 and P0. Androgen receptor was localized to germ and interstitial cells. The effects of methoxychlor, HPTE, estradiol, and testosterone on cell growth of perinatal testes was determined with a thymidine incorporation assay in postnatal day zero (P0) testis cell cultures. Methoxychlor (0.002, 0.02, and 0.2 microM) and HPTE (2 and 20 microM) stimulated thymidine incorporation in P0 testis cell cultures in a similar manner to estradiol (0.01, 0.1, and 1 microM). In addition, testosterone (0.1 microM) also stimulated thymidine incorporation in P0 testis cultures. Observations suggest that methoxychlor and its metabolite HPTE can alter normal embryonic testis development and growth. The actions of methoxychlor and HPTE are likely mediated in part through the steroid receptors confirmed to be present in the developing testis.

Expression and action of neurotropin-3 and nerve growth factor in embryonic and early postnatal rat testis development.

The current study examines the expression and potential actions of neurotropin-3 (NT3), nerve growth factor (NGF), and their receptors during morphological sex determination (seminiferous cord formation) and perinatal rat testis development. The expression of neurotropins and their receptors was analyzed with immunohistochemistry. Cellular localization of neurotropin ligand and receptor proteins changed during embryonic testis development. Neurotropin-3 was localized to Sertoli cells at Embryonic Day 14 (E14), was present in gonocytes at Postnatal Day 0 (P0), and after birth became localized to the interstitium and Sertoli cells (P3-P5). The expression of trk C (the high affinity receptor for NT3) was localized to mesonephric ducts and cells surrounding the cords (E14-E18). In addition, Sertoli cells and preperitubular cells surrounding the cords at E14 also stained for trk C. Neurotropin-3 was expressed in gonocytes and Sertoli cells at P0-P5. Nerve growth factor was detected in Sertoli cells at E14, was clearly in Sertoli and interstitial cells at E16 and E18, and in Sertoli, germ, and interstitial cells from P0-P5. The expression of trk A (the high affinity receptor for NGF) was located in Sertoli and interstitial cells at E16-P5. To determine the actions of neurotropins during embryonic and perinatal testis development, experiments were conducted on E13 and P0 testis. Antisense oligonucleotide experiments with NT3 were used on E13 testis organ cultures to determine effects on seminiferous cord formation. Cord formation was inhibited in 40% of the organ cultures treated with the antisense NT3 oligonucleotides, while no inhibition was observed with sense oligonucleotides. In P0 testis cultures, both NT3 and NGF alone and in combination stimulated thymidine incorporation into DNA. Therefore, the neurotropins are involved in embryonic morphological events (cord formation; NT3) and in growth of the perinatal testis (P0; NT3 and NGF). To define further the growth effects of neurotropins on testis development, expression of transforming growth factor alpha and beta (TGF alpha and TGF beta) were examined in response to neurotropins. The P0 testis cultures were treated with neurotropins, and expression of mRNA for TGF alpha and TGF beta was analyzed utilizing a quantitative reverse transcription-polymerase chain reaction assay. Nerve growth factor and NT3 alone or in combination inhibited expression of mRNA for TGF alpha while NT3 increased mRNA expression of epidermal growth factor receptor. The combination treatment of neurotropins inhibited expression of TGF beta 1 and increase expression of TGF beta 3. In summary, observations suggest that NT3, NGF, trk A, and trk C are localized to cells critical to seminiferous cord formation and appear to be important regulators of morphological sex determination. In addition to these morphological effects, both NT3 and NGF stimulate P0 testis growth and may elicit their action through altering the expression of locally produced growth factors such as TGF alpha and TGF beta. Taken together these results suggest that neurotropins are regulators of paracrine cell-cell interactions that result in morphological sex determination and perinatal testis growth.

Role of transforming growth factor-alpha and the epidermal growth factor receptor in embryonic rat testis development.

Embryonic testis development requires the morphogenesis of cords and growth of all cell populations to allow organ formation. It is anticipated that coordination of the growth and differentiation of various cell types involves locally produced growth factors. The current study was an investigation of the hypothesis that transforming growth factor-alpha (TGF-alpha) is involved in regulating embryonic testis growth. TGF-alpha has previously been shown to function in the postnatal testis. TGF-alpha and other members of the epidermal growth factor (EGF) family act through the epidermal growth factor receptor (EGFR) to stimulate cell proliferation and tissue morphogenesis. To understand the potential actions of TGF-alpha in the embryonic testis, general cell proliferation was investigated. Characterization of cell proliferation in the rat testis throughout embryonic and postnatal development indicated that each cell type has a distinct pattern of proliferation. Germ cell growth was transiently suppressed around birth. Interstitial cell growth was high embryonically and decreased to low levels around birth. A low level of Sertoli cell proliferation was observed at the onset of testis cord formation. Sertoli cell proliferation in early embryonic development was low; the levels were high later in embryonic development and remained high until the onset of puberty. Both TGF-alpha and the EGFR were shown to be expressed in the embryonic and postnatal rat and mouse testis. Perturbation of TGF-alpha function using neutralizing antibodies to TGF-alpha on testis organ cultures dramatically inhibited the growth of both embryonic and neonatal testis. TGF-alpha antibodies had no effect on cord formation. The TGF-alpha antibody was found to be specific for TGF-alpha in Western blots when compared to EGF and heregulin. Testis growth was also inhibited by perturbation of EGFR signaling using an EGFR kinase inhibitor. Therefore, TGF-alpha appears to influence embryonic testis growth but not morphogenesis (i.e., cord formation). Treatment of embryonic testis organ cultures with exogenous TGF-alpha also perturbed development, leading to an increased proliferation of unorganized cells. Testis from EGFR and TGF-alpha knockout mice were analyzed for testis morphology. TGF-alpha knockout mice had no alterations in testis phenotype, while EGFR knockout mice had a transient decrease in the relative amount of interstitial cells before birth. Observations suggest that there may be alternate or compensatory factors that allow testis growth to occur in the apparent absence of TGF-alpha actions in the mutant mice. In summary, the results obtained suggest that TGF-alpha is an important factor in the regulation of embryonic testis growth, but other factors will also be involved in the process.

Role of neurotropins in rat embryonic testis morphogenesis (cord formation).

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 controls or NT3 knockout mice. Combined observations suggest compensation between the different neurotropin ligands, receptors, and/or possibly different growth factors for this critical biological process. In summary, results suggest a novel nonneuronal role for neurotropins in the process of cord formation during embryonic rat testis development. The hypothesis developed is that neurotropins are involved in the progression of male sex differentiation and are critical for the induction of embryonic testis cord formation.

Expression and action of transforming growth factor beta (TGFbeta1, TGFbeta2, and TGFbeta3) during embryonic rat testis development.

The objective of the current study was to determine the role of transforming growth factor beta (TGFbeta) during seminiferous cord formation and embryonic testis development. The expression pattern of mRNA for TGFbeta isoforms was evaluated during testis development through a quantitative reverse transcription-polymerase chain reaction (QRT-PCR) procedure. Expression of mRNA for TGFbeta1 was highest at postnatal day 0 (P0) and P10. In contrast, TGFbeta2 was high at embryonic day 15 (E15), declined at E16, and showed a transient increase at P0 through P3 of testis development. Interestingly, expression of mRNA for TGFbeta3 was high during embryonic development and then declined after P3. Immunohistochemical localization of TGFbeta1 and TGFbeta2 demonstrated expression in Sertoli cells at E14 and in the seminiferous cords at P0. Selective interstitial cells expressed high concentrations of TGFbeta1 and TGFbeta2 in P0 testis. TGFbeta3 was expressed in selective cells at the junction of the E14 testis and mesonephros. The cells expressing TGFbeta3 in the testis appeared to be preperitubular cells that resided around the seminiferous cords. TGFbeta3 was localized to gonocytes in P0 testis. TGFbeta1 was found to have no influence on seminiferous cord formation in embryonic organ cultures of E13 testis. In contrast, growth of both E13 and E14 embryonic organ cultures was inhibited by TGFbeta1 and resulted in reduced testis size (40% of controls) with fewer cords present. A P0 testis cell culture and thymidine incorporation assay were used to directly examine the effects of recombinant TGFbeta1. TGFbeta1 alone had no influence on thymidine incorporation in P0 testis cell cultures when compared to controls. Interestingly, TGFbeta1 inhibited epidermal growth factor (EGF), and 10% calf serum stimulated P0 testis cell growth but not FSH-stimulated growth. Therefore, TGFbeta1 appears to inhibit testis growth in both the embryonic and early postnatal periods. The hormonal regulation of TGFbeta expression was measured using P0 testis cell cultures and a QRT-PCR procedure for each TGFbeta isoform. High concentrations of EGF stimulated expression of mRNA for TGFbeta1 after 24 h but suppressed expression of TGFbeta3. In contrast, there was no effect of FSH on TGFbeta isoform expression. In summary, TGFbeta regulates embryonic and P0 testis growth through inhibiting the actions of positive growth factors such as EGF. In addition, EGF but not FSH appears to regulate TGFbeta isoform expression. Combined observations from the present study demonstrate that TGFbeta isoforms are differentially expressed and appear to be regulators of testis growth during the embryonic and early postnatal periods.

Action of retinoids on embryonic and early postnatal testis development.

The current study investigates the hypothesis that retinoids have a role in embryonic testis development. The action of retinoids on testis development and the expression of retinoic acid receptors (RAR alpha, RAR beta, RAR gamma) were examined. In embryonic day 13 (E13; plug date = E0) testis organ cultures an RAR-selective agonist and all-trans retinoic acid completely inhibited seminiferous cord formation. In contrast, an RAR alpha-selective antagonist had no effect. RT-PCR demonstrated that RAR alpha messenger RNA (mRNA) was expressed at all developmental time points evaluated, which included embryonic day 14 (E14) through postnatal day 30 (P30). Expression of RAR beta mRNA was present at E15 through P2, whereas RAR gamma mRNA was expressed at E18 through P2. Cellular localization of receptors by immunohistochemistry indicated that RAR alpha was localized to the interstitium at E18 and to the seminiferous cords by P0. RAR beta and RAR gamma were detected in both interstitium and cords at E16 and by E18 were mainly expressed in the cords. At P0 RAR beta and RAR gamma were localized to the germ cell populations. To examine retinoid actions, the growth of P0 testis cultures were investigated. Interestingly, retinol and retinoic acid did not inhibit growth of P0 testis cultures but did inhibit the action of growth stimulators. Retinoic acid inhibited FSH, EGF, and 10% calf serum stimulated growth in P0 testis cultures. The hypothesis tested was that the inhibitory effects of retinoids on P0 testis growth may be mediated through the growth inhibitor, transforming growth factor-beta (TGF beta). The action of retinoids on TGF beta mRNA expression was examined in P0 testis cultures. Retinoic acid stimulated TGFbeta3 mRNA expression within 24 h and increased expression of TGFbeta1 and TGFbeta2 after 72 h. Retinol increased expression of TGFbeta1 and TGFbeta2 but not TGFbeta3 after 72 h of treatment. These observations indicate that retinoic acid can influence seminiferous cord formation and testis growth. The inhibitory actions of retinoids may in part be mediated through increased expression of TGFbeta isoforms.

Developmental and hormonal regulation of transforming growth factor-beta1 (TGFbeta1), -2, and -3 gene expression in isolated prostatic epithelial and stromal cells: epidermal growth factor and TGFbeta interactions.

Growth factors are postulated to mediate stromal-epithelial interactions in the prostate to maintain normal tissue physiology. Transforming growth factor-beta (TGFbeta) has been shown to influence the prostate and probably mediate stromal-epithelial interactions. TGFbeta1 messenger RNA (mRNA) expression is stimulated after castration and can be suppressed by in vivo treatment with androgens. The hypothesis tested is that TGFbeta is regulated not only by androgen, but also by a network of locally produced growth factors that influence prostatic growth and differentiation. Epithelial and stromal cells from 20-day-old rat ventral prostate were isolated and used to test this hypothesis. The expression of mRNA for TGFbeta1, -2, and -3 was analyzed by a quantitative RT-PCR procedure. Observations from this assay demonstrate that both epithelial and stromal cells express the mRNA for TGFbeta1, -2, and -3. TGFbeta1 mRNA expression was constant during development of the prostate. TGFbeta2 mRNA expression was elevated at birth, then declined and elevated again at 100 days of age. TGFbeta3 mRNA expression was high during puberty and young adult ages then declined at 100 days of age. TGFbeta2 and TGFbeta3 expression are inversely related during prostate development. After castration of 60-day-old rats, both TGFbeta1 and TGFbeta2 mRNA were enhanced. Interestingly, TGFbeta3 mRNA was significantly suppressed after castration. Epidermal growth factor (EGF) stimulated TGFbeta1 mRNA expression in stromal cells (6-fold increase), whereas keratinocyte growth factor stimulated TGFbeta2 mRNA in epithelial cells. TGFbeta inhibited both testosterone- and EGF-stimulated prostatic stromal and epithelial cell growth. EGF and TGFbeta also inhibited prostatic ductal morphogenesis and growth in organ culture. Immunocytochemical localization of TGFbeta in 20-day-old prostate demonstrated predominately stromal localization of the protein. These results indicate that the isoforms of TGFbeta2 and TGFbeta3 are differentially regulated during prostate development, suggesting distinct regulatory mechanisms. Testosterone did not affect TGFbeta expression in cultured prostatic cells. These observations suggest that the in vivo effects of castration on TGFbetas are regulated indirectly through a complex network of growth factors, not simply by direct androgen depletion. The ability of EGF to inhibit prostatic ductal morphogenesis and growth in organ culture is postulated to be in part mediated by the increase in TGFbeta1 expression. In summary, a network of growth factor-mediated stromal-epithelial interactions is needed to maintain prostate growth and development. TGFbeta is postulated to have an important role in this process.

Concentrations of gonadotropins, estradiol and progesterone in sows selected on an index of ovulation rate and embryo survival.

The objective of this study was to determine concentrations of follicle stimulating hormone (FSH), luteinizing hormone (LH), progesterone (P4) and 17beta-estradiol (E2) in sows from a line selected on an index which emphasized ovulation rate (Select) and from a control line. A further classification of the sows in each line was made according to the estimated number of ovulations during an estrous cycle. Sows in the Select line were ranked into a high (HI) or low group (LI) when their estimated number of ovulations were 25 or more and 14 to 15, respectively. Sows of the control line were classified into groups as high (HC) or low (LC) when the estimated values for ovulation rate were 14-15 and 8-9 ovulations, respectively. Blood samples were collected every 12 h during a complete estrous cycle and samples were analyzed for concentrations of FSH and LH. Samples collected every 24 h were assayed for P4 and E2. Mean concentrations of FSH, LH, P4 and E2 did not differ (P > 0.10) between lines or between HI and LI or HC and LC groups. Selection of pigs for ovulation rate and embryonal survival did not affect concentrations of FSH, LH, P4 and E2 in sows during the estrous cycle.

Role of basic-helix-loop-helix transcription factors in Sertoli cell differentiation: identification of an E-box response element in the transferrin promoter.

Sertoli cells are critical for testicular function and maintenance of the spermatogenic process. The induction of Sertoli cell differentiation in the embryo promotes testicular development and male sex determination. The progression of Sertoli cell differentiation during puberty promotes the onset of spermatogenesis. The maintenance of optimal Sertoli cell differentiation in the adult is required for spermatogenesis to proceed. The current study was designed to investigate the transcriptional regulation of Sertoli cell differentiation through the analysis of a previously identified marker of differentiation, transferrin gene expression. Sertoli cells produce transferrin to transport iron to developing spermatogenic cells sequestered within the blood-testis barrier. The transferrin promoter was characterized and found to contain two critical response elements, designated Sertoli element 1 (SE1) and Sertoli element 2 (SE2). Through sequence analysis, SE2 was found to contain an E-box response element, which has been shown to respond to basic-helix-loop-helix (bHLH) transcription factors. The bHLH proteins are a class of transcription factors associated with the induction and progression of cell differentiation. bHLH proteins dimerize through the conserved helix-loop-helix region and bind DNA through the basic region. Nuclear extracts from Sertoli cells were found to cause an E-box gel shift when the cells were stimulated to differentiate in culture, but not under basal conditions. The SE2 gel shift of Sertoli nuclear extracts was competed with excess unlabeled SE2 or E-box DNA fragments. Several Sertoli nuclear proteins associate with the SE2 gel shifts, including 70-, 42-, and 25-kDa proteins. Therefore, the critical SE2 element in the transferrin promoter is an E-box element capable of binding bHLH transcription factors. The ubiquitously expressed E12 bHLH protein dimerizes with numerous cell-specific bHLH factors. A Western blot analysis demonstrated that E12 was present in Sertoli cell nuclear extracts and associated with the SE2 gel shift. A ligand blot of Sertoli cell nuclear extracts with radiolabeled E12 had apparent bHLH proteins when the cells were stimulated to differentiate. The E-box sequence in the SE2 fragment of the transferrin promoter was CATCTG and was similar in gel shifts to the consensus E-box elements (CANNTG) previously characterized. A bHLH inhibitory factor (Id) competed and inhibited formation of the Sertoli cell nuclear extract E-box gel shift. To extend this observation, Id protein was overexpressed in cultured Sertoli cells. A transferrin promoter chloramphenicol acetyltransferase construct was used to monitor Sertoli cell function. The presence of Id suppressed the activation of the promoter induced by Sertoli differentiation factors. Therefore, the inhibition of Sertoli bHLH factors by Id suppressed Sertoli cell differentiated function, as measured by transferrin expression. An E-box-chloramphenicol acetyltransferase construct was also found to be active in Sertoli cells when cells were induced to differentiate. Screening the computerized nucleotide data bases demonstrated that putative E-box response elements are present in the promoters of a large number of Sertoli cell differentiated genes. In summary, a critical E-box response element has been identified in the transferrin promoter that can be activated by bHLH factors (e.g. E12) present in Sertoli cells. Inhibition of Sertoli bHLH factors by Id suppresses Sertoli cell differentiated function (i.e. transferrin expression), suggesting that bHLH transcription factors may be important in regulating Sertoli cell differentiated functions.