Expression of oestrogen receptor alpha during development of the skeleton in mice fetuses: immunohistochemical study

Sequential pattern of ossification and expression of oestrogen receptor alpha (ERalpha) during development of the skeleton in male and female mice fetuses was investigated. Twenty-seven mice fetuses of gestational age between 14.5 and 18.5 days post coitum (p.c.) were examined by haematoxylin-eosin and toluidine blue staining to determine the ossification. The presence of ERalpha was detected by immunostaining using ERalpha-specific antibodies. Ossification centres were determined in fetuses of 14.5 days p.c. of both sexes in the base of skull, ribs and front limbs, while in the mandible ossification was observed only in female fetuses at that age. ERalpha was found in all investigated tissues in which the occurrence of ossification centres was determined. ERalpha was first detected in some tissues involved in ossification at 14.5 days p.c. in fetuses of both genders. There were some minor gender differences in the pattern of ERalpha expression. ERalpha was localized in the metatarsal chondrogenic condensations at 14.5 days p.c. and in phalangeal chondrocytes at 17.5 and 18.5 days p.c. only in females. ERalpha-positive osteogenic cells at 14.5 days p.c. in the mandible were seen only in females. At 16.5 days p.c. male but not female fetuses expressed ERalpha in the vertebrae. Our findings support the view that ERalpha protein is found in the tissues that undergo bone formation and that ERalpha expression in these tissues shows only minor gender differences in mice fetuses.     

Neuroendocrine Regulation of Growth Hormone and Luteinizing Hormone in Fetal and Neonatal Pig

Ontogeny of the production and the regulation of growth hormone and Luteinizing hormone is studied in a series of experiments utilizing male and female pigs at different fetal and neonatal stages. Growth hormone mRNA is detectable in both sexes as early as d. 50 p.c. The mRNA levels increase to reach the maximum levels at d. 95–110 p.c. Plasma levels of GH follow the developmental patterns of GH mRNA. A sex difference is evident around d. 80–90 p.c. with males having higher GH levels than females. The stimulatory but not the inhibitory mechanisms of GH secretion are fully functioning in the pig fetus. LHβ mRNA is detectable earlier in females (d. 50 p.c.) than in males (d. 65 p.c.). Plasma concentrations of LH increase with fetal age in female fetuses, but in male fetuses there is no distinct developmental pattern evident. Basal LH secretion achieves maximum levels in both sexes after birth. Opioids do modulate fetal LH secretion, however, the mode of their action is age-dependent.     

Possibility of Neospora caninum infection by venereal transmission in CB-17 scid mice

CB-17 scid and BALB/c male mice were inoculated intraperitoneally with Neospora caninum to examine the possibility of its venereal transmission. Some of these mice were killed on days 7 and 20 post-inoculation to examine the genital organs for presence of the parasite. The remaining scid male mice were housed with non-infected female mice from day 7 p.i. and kept with them for 14 days. These scid mice died between days 28 and 35 p.i. N. caninum DNA was detected in the testis of mice on days 7 and 20 p.i. by PCR and tachyzoite viability was determined by bioassay conducted by means of mouse inoculation. Microscopically, fewer tachyzoites were detected in the testis obtained on day 20 p.i., than in other organs. The inoculated BALB/c male mice survived until the end of the experiment with no clinical signs and N. caninum DNA was detected in the testis on day 7 p.i. but not on day 14 p.i. Five of eight female scid mice housed with infected males became pregnant. Tachyzoites were detected in three of these mice and their neonates (n=3, 5 and 13, respectively). In three non-pregnant mice, no parasite was detected. Two of the four female BALB/c mice housed with infected male scid mice became pregnant but the parasite was not detected in them or in the neonates (n=3 and 13, respectively). These results indicate that the tachyzoites were present in the genital organs of the immunodeficient mice from day 7 p.i. and suggest that transmission may occur through mating with male mice.     

Relationship of fetal position within the uterus to fetal weight, placental weight, testosterone, estrogens, and thymosin beta 4 concentrations at 70 and 104 days of gestation in swine.

Fetal intrauterine position relative to the sex of adjacent fetuses has an effect on reproductive performance in rodents. An experiment was conducted to determine whether sex of adjacent fetuses in utero has an influence on fetal and placental weights and whether the hormonal mechanisms documented in rodents are similar in fetal pigs. Sows were slaughtered at 70.1 +/- 1.7 d (n = 123) and 104.5 +/- .05 d (n = 135) of gestation. The fetuses and placentas were removed from the uterus and the position and sex of each fetus was recorded to indicate whether the fetus was between two males, two females, or a male and a female. Fetal blood was sampled for later hormonal analysis. At 70 d of gestation, male fetal and placental weights were heavier than those of females (P less than .05), but no differences were detected relative to the sex of adjacent fetuses. At 104 d of gestation, a fetus surrounded on each side in utero by fetuses of the opposite sex (two males or two females) was lighter in weight than a fetus surrounded by fetuses of the same sex (P less than .01). Differences in fetal weight due to the sex of adjacent fetuses were not related to placental function because placental weights were generally not different at 104 d of gestation. By 104 d of gestation, most placentas were not separated by necrotic regions and were in close apposition with surrounding placentas. No differences in growth or development could be related to hormonal effects (testosterone, estrone, or estrone sulfate) from surrounding fetuses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intrauterine position effects in male and female swine: subsequent survivability, growth rate, morphology and semen characteristics

Pigs of known intrauterine position were obtained from 31 litters by a procedure in which donor sows were slaughtered at d 112 of gestation, their uteri removed and piglets delivered manually. Uterine position was recorded for each piglet as being positioned between two female fetuses (OM), between a male and female fetus (1M), between two male fetuses (2M), between a female fetus and the tip of the uterine horn (OE) or between a male fetus and the tip of the horn (1E). Piglets were fostered as litter groups to recipient sows and reared in these groups until 120 d of age. They then were regrouped and housed as groups of three and six for males and females, respectively. Intrauterine position had no effect on birth weight or survivability of pigs of either sex, although pigs positioned in utero nearest the ovaries (OE and 1E) tended to be heavier at birth. Body weights were similar among groups in each sex at 120 and 175 d of age when given ad libitum access to feed; however, 2M males gained more weight from d 175 to 270 under restricted feeding conditions (P less than .05). Intrauterine position had no effect on anogenital distances either at birth or 120 d of age, and predicted testes weights were similar among males from different positions. Semen characteristics at 220 d of age did not appear to vary due to prenatal environment. Although volume tended to be less for 0M males (P less than .12), concentration, motility and sperm/ejaculate were similar among groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Correlated changes in reproductive components accompanying 10 generations of selection for improved sow productivity index.

Reproductive components were compared between a line of sows selected (S) for improved sow productivity index (SPI = 6.5 x number born alive + adjusted 21-d litter weight) and sows from an unselected control (C) line. Generation 9 and 10, second-parity, Landrace sows were chosen from both the S (n = 35) and C (n = 33) line. Sows were slaughtered at a commercial slaughter plant at approximately 75 d of gestation and their reproductive tracts were recovered. Reproductive tracts were evaluated for uterine weight (UTWT), uterine horn length (UTLN), ovulation rate (OR), number of fully formed fetuses (NF), number of mummified fetuses (NM), percentage of fetal survival (FS = NF/OR), fetal space (FSPACE = UTLN/[NF + NM]), and fetal position, sex, and weight. Select-line sows had greater NF (P less than .10) and higher FS (P less than .10) than C-line sows. Select-line sows had longer (P less than .05), and heavier (P less than .01) uteri than C-line sows. However, uterine length adjusted for NF was not different between the two lines. Uterine weight adjusted for NF was greater in S-line sows (P less than .05). Select-line sows had greater total fetal weight (TFWT) (P less than .05) than did C-line sows. Female fetuses positioned between two male fetuses were lighter in weight than all other female fetuses (P less than .01). Male fetuses positioned between two female fetuses did not differ in weight from all other male fetuses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Observations on the Fetal Morphology in Myocastor Coypus Bonariensis (Coypu) (Rodentia,Myocastoridae)

The goal of this work was to characterize the morphology of fetuses of Myocastor coypus bonariensis (coypu) after 60, 90, 120 and 135 days post‐coitus (d.p.c.). At all the ages, gestational sacs showed an elliptical shape. Placentas were discoidal, with a unilobular external appearance in fetuses of 60 d.p.c. and with lobulations separated by a groove in fetuses of other ages. The umbilical cord, wrapped around the fetal body, showed a proximal dilatation in 60‐d.p.c. fetuses, being its course uniform and a little spiral in the other ages. The 60‐d.p.c. fetuses showed differentiation of corporal regions, prominent encephalic vesicles and little developed earlobes located in the laterals of the skull, in the limit with the cervical area and in straight line with the optical vesicles. The primitive forelimbs showed differentiation of arm, forearm and hand. In the hindlimbs, the inter‐digital membrane was observed joining the fingers along all their length. Fetuses of 90 d.p.c. showed developed eyelids and closed palpebral cleft, developed auricular pavilions (earlobes), hairy follicles in the facial region (vibrissae) and in the skull and bent limbs. Fetuses of 120 and 135 d.p.c. showed a bodily cover constituted by a dense pilous covering, spread palpebral clefts, open mouth, bent limbs and inter‐digital membranes in the hindlimbs.     

Morphological changes of physeal cartilage and secondary ossification centres in the developing femur of the house mouse (Mus musculus): A micro‐CT based study

In mammals, long bones are formed by ossification of a cartilaginous mould during early stages of development, through the formation of structures called the primary ossification centre, the secondary ossification centres (SOCs) and the physeal cartilages (PCs). The PC is responsible for long bone growth. The morphology of the PC and the SOCs varies during different stages of femoral growth. In this respect, several details involving the process of murine femoral development are lacking. In the present study, a morphological characterization of femur development from the embryonic period to adulthood in mice was studied using micro‐computed tomography (micro‐CT). To achieve this aim, femora were collected at embryonic day (E) 14.5, E16.5 and E18.5 and at postnatal day (P)1, P7, P14, P35, P46 and P52. CT images were obtained using a micro‐CT scanner (X‐SkyScan 1172; Micro Photonics) and analysed using the micro‐CT 3D visualization software Mimics (Materialise NV, Leuven, Belgium) and NRecon (Micro Photonics). The results of the present study revealed that the femur and its PCs and SOCs undergo morphological changes during different stages of development, including changes in their shape as well as position and thickness. These changes may be due to the response of the femur to mechanical loads imposed by muscle surrounding the bone during these stages of development. The result of the present study is important to improve our knowledge related to ossification and growth patterns of mouse femur during development.     

胚胎期大鼠性腺生长与分化的研究

本试验旨在研究胚胎期大鼠性腺生长与分化的情况。选取12.5~15.5 dpc SD大鼠胚胎为研究对象,运用PCR技术进行大鼠胚胎性别鉴定,采用H-E技术对大鼠性腺分化形态进行观察。结果表明:12.5 d的鼠胚肾管已经开始形成,生殖嵴已经建立,此时仍无明显的性别分化形态;13.5 d的鼠胚开始出现性别分化的迹象,雄性的原始性索开始形成,雌性性腺分化比雄性稍晚,此期仍不易辨别出典型的卵巢特征结构;14.5 d的鼠胚性腺形态初步成型,此期性别明显分化,雄性的原始性索开始分化为实心原始生精小管,雌性胚胎中性腺分为两层,初步形成卵巢特征;15.5 d的鼠胚,雄性胚胎性腺中已经具有明显的曲精小管的雏形,雌性胚胎卵巢特征也开始明显。大鼠胚胎性腺从13.5 d胚龄时开始分化,15.5 d胚龄性腺特征明显。     

Sexual differentiation in litter-bearing mammals: influence of sex of adjacent fetuses in utero

In rodents and swine, individual differences in a broad range of characteristics correlate with intrauterine position during fetal life. By identifying the intrauterine position of mice at cesarean delivery, we can predict reliably postnatal reproductive traits such as genital morphology, timing of puberty, length of estrous cycles, timing of reproductive senescence, sexual attractiveness, sexual behavior, aggressiveness, daily activity level, body weight and tissue enzyme activity in females; in males we can predict genital and brain morphology, sexual behavior, aggressiveness, daily activity level, body weight, and tissue enzyme activity. In mice, as in all mammals, male fetuses have greater concentrations of testosterone than do females. In addition, female mouse fetuses have greater circulating concentrations of estradiol than do male fetuses, a condition not found in all mammals. A mouse fetus positioned between males has greater concentrations of testosterone than does a fetus of the same sex positioned between females, and a fetus positioned between females has greater concentrations of estradiol than does a fetus of the same sex positioned between males. Gonadal steroids regulate differentiation of secondary sexual characteristics. Studies in which the effects of intrauterine position have been eliminated by exposing fetuses to steroid receptor blockers reveal the critical role of steroids in mediating this phenomenon. The intrauterine position phenomenon provides the only mammalian model for relating postnatal traits to concentrations of endogenous hormones to which individuals are exposed during fetal life. Results from studies using this naturally occurring experimental system in litter-bearing species have given insights concerning the consequences of individual differences in steroid concentrations during sexual differentiation that likely apply to all mammals. One specific hypothesis is that circulating estradiol may interact with testosterone in mediating some aspects of sexual differentiation in rodents and, thus, possibly in other mammals.     

Factors relating to urine spraying and fighting in prepubertally gonadectomized cats

The fact that male cats are routinely castrated prior to full sexual maturity for behavioral reasons prompted a survey of the prevalence of these behaviors in prepubertally gonadectomized male and female cats. About 10% of the 134 male and 5% of the 152 female cats studied engaged in spraying on a frequent basis later in adult life. Problem spraying and fighting in males was of the same order of magnitude as in males castrated in adulthood after the problem behaviors had appeared. The age of prepubertal castration, ranging from 6 to 10 months, had no influence on spraying or fighting later in life. It was found that male cats are more likely to spray and fight if they are in households with female cats than with other male cats. With regard to female cats, spraying or fighting was less than that of male cats. Age at the time of gonadectomy was not a factor in their tendency to spray or fight. Possible prenatal masculinization of female fetuses by male fetuses, as revealed by littermate composition, was not a factor in the predisposition of female cats to spray or fight.     

Vertical Transmission of Brucella abortus Causes Sterility in Pregnant Mice

The mechanisms of abortion and sterility induced by bacterial infection are largely unknown. In the present study, we found that Brucella abortus, a causative agent of brucellosis and facultative intracellular pathogen, caused sterility in pregnant mice. We have recently established a mouse model for abortion induced by B. abortus infection and high rates of abortion are observed for bacterial infection on day 4.5 of gestation, but not for other days. Infected newborn (first generation) mice showed poor growth compared with uninfected newborn mice and bacterial replication in the spleen of the former was observed over a long period. When infected first generation female mice were mated to infected first generation male mice, the number of fetuses was significantly less than that in uninfected first generation mice. These infected second generation mice also showed poor growth. These results suggest that vertical transmission of B. abostus causes sterility in pregnant mice and our mouse model would be useful for the investigating of brucellosis.     

Demonstration of microchimerism in pregnant sows and effects of congenital PRRSV infection

ABSTRACT: The presence of foreign cells within the tissue/circulation of an individual is described as microchimerism. The main purpose of the present investigation was to study if microchimerism occurs in healthy sows/fetuses and if porcine reproductive and respiratory syndrome virus (PRRSV) infection influences this phenomenon. Six dams were inoculated intranasally with PRRSV and three non-inoculated dams served as controls. Male DNA was detected in female fetal sera of all dams via PCR. Male DNA was also detected in the maternal circulation. Sex-typing FISH showed the presence of male cells in the female fetal organs and vice versa. PRRSV infection did not influence microchimerism, but might misuse maternal and sibling microchimeric cells to enter fetuses.     

Changes in pelvic conformation and peripheral estrone concentrations in pre- and post-partum beef cows

An experiment was conducted to investigate the temporal relationship of peripheral estrone (E1) concentration to changes in the size of the pelvic opening preceding and immediately following parturition. Twenty-six multiparous beef cows were observed from approximately 50 d prepartum to 7 d postpartum. Blood samples were collected at 7 d intervals preceding calving and at 1, 3 and 7 d following for E1 quantitation. Estimates of pelvic opening area were made at the time of blood sampling. Peripheral E1 concentrations were elevated beginning at approximately 25 d prepartum. Dams bearing male fetuses had greater (P less than 0.01) concentrations of E1 than did dams with female fetuses. Calf birth weight was correlated (r = 0.44, P less than 0.01) with E1 levels from 10 d prepartum through parturition. Postpartum pelvic area was greater for cows giving birth to male calves, with no significant differences for calf birth weights by sex. Correlations were observed between E1 concentration, and pelvic area measured from 50 d prepartum to 7 d postpartum (r = 0.26, P less than 0.01), 10 d prepartum to calving (r = 0.42, P less than 0.01), and from calving to 7 d postpartum (r = 0.33, P less than 0.01). Percentage increase in E1 concentration from 50 d prepartum to calving was significantly correlated (r = 0.75, P less than 0.01) to percentage pelvic area increase over the same period. A correlation also exists between maternal E1 concentrations and fetal sex and pelvic area. In summary, the increased estrogen concentrations in cows with male calves may facilitate pelvic spread, resulting in a larger pelvic opening.     

H-Y单抗检测方法初探

优化间接ELISA条件 ,提高灵敏度 ,建立检测H Y抗体的检测方法。利用BALB/c公鼠免疫同系母鼠 ,制备H Y单抗。PCR验证间接免疫荧光法鉴定胚胎性别准确率。结果表明 :鉴定雄性胚胎的准确率为 83% ( 1 5 / 1 8)。鉴定雌性胚胎的准确率为 94 % ( 1 5 / 1 6)。     

Ossification and Growth Rates of the Limb Long Bones during the Prehatching Period in the Quail (Coturnix coturnix japonica)

The timing of ossification and the growth of six long bones of the prehatching period in the quail was studied. Ninety-nine quail eggs were incubated and in total nine fetuses were selected daily from the sixth to the sixteenth day of incubation. The fetuses were stained with alizarine and alcian blue double colouration. The fetuses were studied under the stereoscopic microscope and linear measurements were obtained from the humerus, ulna, radius, femur, tibia and fibula. The first appearance of the primary ossification centres in the diaphysis of the studied bones was found to occur between the sixth and the seventh day of incubation. Different growth patterns between the bones of the leg and of the wing were observed. Humerus and tibia showed the greatest growth rate while the radius and fibula showed the lowest.     

Influence of prenatal testosterone treatment on foetal and prepubertal LHbeta-subunit mRNA and plasma LH concentrations in the female pig

The aim of this study was to investigate the potential role of foetal androgens in determining the sexual dimorphism in LH gene expression. Starting on day 30 p.c. pregnant sows were treated i.m. with testosterone propionate (TP) three times at 2-day intervals (TP30 treatment) or received additional TP treatment starting on day 40 p.c. (TP30/40). Sows were allowed to farrow and after frequent blood samples for LH determination were collected prepubertally (6 months) from the female offspring anterior pituitary LHbeta subunit mRNA levels were determined. In Experiment 2 pregnant sows were treated as TP30 before or received similar treatment starting on day 40 p.c. (TP40), but anterior pituitary LHbeta mRNA and plasma LH concentrations were determined at day 80 p.c. TP30 or TP30/40 treatment did not affect mean plasma LH concentrations nor LHbeta mRNA levels at 6 months of age but caused marked masculinization of external genitalia. At day 80 p.c. LHbeta mRNA and plasma LH levels were higher in female than in male foetuses. TP40 treatment suppressed LHbeta mRNA and plasma LH levels while TP30 treatment had no effect on LHbeta mRNA levels but caused masculinization of external genitalia in contrast to TP40. Our findings support the notion that the peak in plasma testosterone observed by others in the male pig foetus 5 weeks p.c. not only determines sexual differentiation of the LH surge mechanism but also LH gene expression in the foetus. The critical period for this process seems to succeed phenotypic differentiation (which appears to be largely completed before day 40 p.c.). The tonic mode of prepubertal LH gene expression and LH secretion in female pigs is not affected greatly by testosterone treatment at the stages of development that were investigated.     

昆明小鼠不同性别早期胚胎体外培养发育潜力观察

自主设计2对引物,提取已知性别小鼠肝细胞DNA,扩增雄性小鼠特有的Sry基因及雌、雄小鼠共有的ZFX基因,建立双重PCR性别鉴定方法.提取小鼠早期8细胞胚胎单卵裂球、双卵裂球DNA,分别进行双重PCR性别鉴定,选择出早期8细胞胚胎的适宜模板量;将已测性别的8细胞胚胎按照雌、雄性别分开培养,观察统计雌、雄8细胞胚胎发育至囊胚的比率.试验结果显示,与单个卵裂球DNA模板量相比,双卵裂球的模板量检出率高,两者之间差异显著(75.00%Vs 93.33%,P<0.05);经过性别鉴定的雄性8细胞胚胎比雌性8细胞胚胎发育至囊胚的比率高,两者之间差异显著(53.33%Vs 33.33%,P<0.05).结果表明,采用双重PCR性别鉴定方法,以双卵裂球的DNA量为模板能够有效的对小鼠早期8细胞胚胎进行性别鉴定;雄性胚胎在体外培养条件下比雌性胚胎具有更好的发育潜力.