Birth of viable puppies derived from breeding cloned female dogs with a cloned male

Since the establishment of production of viable cloned dogs by somatic cell nucleus transfer, great concern has been given to the reproductive abilities of these animals (Canis familiaris). Therefore, we investigated reproductive activity of cloned dogs by (1) performing sperm analysis using computer-assisted sperm analysis and early embryonic development, (2) assessing reproductive cycling by measuring serum progesterone (P4) levels and performing vaginal cytology, and (3) breeding cloned dogs using artificial insemination. Results showed that most parameters of sperm motility in a cloned male dog were within the reference range, and in vivo–matured oocytes from a noncloned female were successfully fertilized by spermatozoa from a cloned male dog and develop normally to the 8-cell stage. Three cloned female dogs displayed normal patterns of P4 levels and morphologic changes of the vaginal epithelium. Two cloned female dogs became pregnant using semen from a cloned male dog and successfully delivered 10 puppies by natural labor. In conclusion, these data demonstrated that both cloned male and female dogs are fertile, and their puppies are currently alive and healthy with normal growth patterns.     

Generation of viable embryos and embryonic stem cell-like cells from cultured primary follicles in mice

Primary follicles retrieved from B6CBAF1 prepubertal mice were cultured in a stepwise manner in an alpha-minimum essential medium-based medium to generate viable embryos and embryonic stem cell (ESC)-like cells. A significant increase in follicle growth and oocyte maturation accompanied by increased secretion of 17beta-estradiol and progesterone was achieved by exposing primary follicles to 100 or 200 mIU of follicle-stimulating hormone (FSH) during culture. More oocytes developed into blastocysts following in vitro fertilization (IVF) or parthenogenetic activation after culture with 200 mIU of FSH during the entire culture period than with 100 mIU. Eleven ESC-like cell lines, consisting of four heterozygotic and seven homozygotic phenotypes, were established from 25 trials of primary follicle culture combined with IVF or parthenogenetic activation. In conclusion, primary follicles can potentially yield developmentally competent oocytes, which produce viable embryos and ESC-like cell lines following in vitro manipulation. We suggest a method to utilize immature follicles, which are most abundant in ovaries, to improve reproductive efficiency and for use in regenerative medicine.     

Biosynthesis of neutral glycosphingolipids in kidney slices from male and female mice

Previous reports from our laboratory (1981. J. Biol. Chem. 256: 13112-13120 and 1983. Endocrinology. 113: 251-258) showed the absence of Nfa-GalCer and Nfa-GaOse2Cer in kidneys of several strains of female mice. These lipids are always present in male kidneys and several other glycolipids are also elevated in males. To test whether this phenomenon is due to lowered biosynthesis in females, glycosphingolipid formation was assessed in kidney slices with [3H]galactose as precursor. The glycolipids were extracted after various incubation periods (from 30 min to 90 min) and individual glycolipids were separated and quantitated by high performance liquid chromatography and radioactivity was determined. The rate of formation of hydroxy fatty acid-containing galactosylceramide was the same in both sexes. The glycolipids which were low or not detectable in female kidney, Nfa-GalCer, Nfa-GaOse2Cer and Hfa-GaOse2Cer were rapidly labeled in the male kidney slices. These results suggest that nonhydroxy fatty acid-containing ceramide:UDP-Gal galactosyltransferase and hydroxy fatty acid-containing galactosylceramide:UDP-Gal galactosyltransferase have elevated activities in males. While the glucosylceramides are labeled at the same rates in both sexes, lactosylceramide appears to be labeled at higher rates in the male tissue. This suggests that glucosylceramide:UDP-Gal galactosyltransferase also has elevated activity in males. In addition, these data show that monohexosylceramides with different ceramide compositions are labeled at different rates.     

Androgen sulphate formation in male and female mice

1. After the administration of large doses of androsterone, epiandrosterone, dehydroepiandrosterone and testosterone to mice, females excreted more of the dose conjugated with sulphuric acid than did males. 2. Liver slices from female mice conjugated androgens with sulphuric acid to a greater extent than did slices from males. 3. Sulphotransferase preparations from livers of female rats and mice catalysed the formation of dehydroepiandrosterone sulphate at a faster rate than preparations from livers of the male animals. 4. A possible explanation for the observed sex differences is discussed.     

Dietary fats influence 'open-field' behaviour of male and female laboratory mice

Swiss mice of differing ages (juvenile and adult) and sexes were fed four specially formulated, pelleted diets containing respectively 8% saturated vegetable fat, 8% soya oil, 8% olive oil and 2% soya oil (with identities hidden from the experimenter) or a local commercial chow (3% crude fat) for 3 or 6 weeks. Subjects were individually housed and were assessed under red lighting for behaviour in a modified 'open field' (a 30 x 20 cm box with a black floor). Videotaped records were analysed using 'The Observer' system, quantifying transitions between inner and outer zones, rearing, freezing, grooming and defaecation as well as location in the two equal-sized zones. Clearly, these non-isocaloric diets differed in palatability, producing complex effects on growth as well as physiological and behavioural measures. Many indices were influenced by age, sex, and the duration of dietary exposure. Interactions between factors were common. Defaecation does not seem to provide a useful index of 'emotionality' in this type of study and investigations lacking a wide range of indices seem unlikely to provide unequivocal support for postulated links between dietary lipids and behaviour. The study broadly supports the contention that dietary fats subtly influence mood in mice.     

Ornithine decarboxylase antizyme in kidneys of male and female mice.

Antizyme, a protein inhibitor of ornithine decarboxylase (ODC), was shown to be induced in mouse kidney by repeated injection of putrescine. Antizyme was also present as a complex with ODC in the kidney of untreated mouse. The amount of the renal ODC-antizyme complex was 3-fold higher in male mice than in female mice. On the contrary, the proportion of ODC present as a complex with antizyme was 24-fold higher in females than in males, and the decay of renal ODC activity after cycloheximide treatment was about 5-fold more rapid in females than in males. Administration of testosterone to female mice, a procedure known to prolong the half-life of renal ODC, increased both ODC activity and the content of ODC-antizyme complex, but decreased the antizyme/ODC ratio in the kidney. These results are consistent with the previous observation in HTC cells that the decay rate of ODC activity in the presence of cycloheximide correlated well with the proportion of ODC present as a complex with antizyme, suggesting the ubiquitous role of antizyme in ODC degradation.     

Time-course of Giardia muris infection in male and female immunocompetent mice

The time-course of acute Giardia muris infection was compared in male and female immunocompetent BALB/c mice that had not previously been exposed to the parasite. No sex-related difference was observed in the time-course of the infection in these mice. Sexually mature mice of both sexes excreted substantial numbers of G. muris cysts (greater than 10(4)/2 hr) over a longer period than did sexually immature mice.     

Multiple activation of chloroform in kidney microsomes from male and female DBA/2J mice

Microsomes from the renal cortex of DBA/2J mice can metabolize chloroform through oxidative and reductive pathways, similar to hepatic microsomes. The oxidative or reductive nature of CHCl₃ activation is strictly dependent on the oxygenation of the incubation mixture, as indicated by the formation of qualitatively different adducts to phos-pholipids (PLs). The protein and lipid binding levels measured in kidney microsomes from control females differed significantly from the binding levels observed with kidney microsomes from male and testosterone-treated female DBA/2J mice in aerobic conditions only. Therefore, the sex-dependent CHCl₃-induced acute nephrotoxicity seems related only with the oxidative CHCl₃activation. The levels of adducts to PL polar heads and to protein showed a strict correlation with each other. Therefore, the assay of adducts to PL polar heads may be used as a substitute for the assay of adducts to protein. This might be especially convenient when studying the effects of both phosgene and the trichloromethyl radicals.     

High-frequency generation of viable mice from engineered bi-maternal embryos

Mammalian development to adulthood typically requires both maternal and paternal genomes, because genomic imprinting places stringent limitations on mammalian development, strictly precluding parthenogenesis. Here we report the generation of bi-maternal embryos that develop at a high success rate equivalent to the rate obtained with in vitro fertilization of normal embryos. These bi-maternal mice developed into viable and fertile female adults. The bi-maternal embryos, distinct from parthenogenetic or gynogenetic conceptuses, were produced by the construction of oocytes from fully grown oocytes and nongrowing oocytes that contain double deletions in the H19 differentially methylated region (DMR) and the Dlk1-Dio3 intergenic germline-derived DMR. The results provide conclusive evidence that imprinted genes regulated by these two paternally methylated imprinting-control regions are the only paternal barrier that prevents the normal development of bi-maternal mouse fetuses to term.     

Effects of female urine on social investigation in male mice

In experiments where female mouse urine or water was introduced on a Perspex block into a test arena, to which a group of male mice familiar with each other had been habituated, female urine was shown to cause a significant increase in the incidence of the social behaviour patterns: nose, investigate, ano-genital sniff and follow. The duration of ano-genital sniff was also significantly greater in the presence of female urine.     

Prior intrauterine position influences body weight in male and female mice

In two longitudinal studies, intrauterine location of male and female Rockland-Swiss mice relative to fetuses of the same and opposite sex dramatically influenced body weight. In one study, body weight of males and females that were located in utero between two female fetuses (OM animals) or between two male fetuses (2M animals) was assessed from birth to the time of weaning (25 days of age). The body weights of 2M females were indistinguishable from those of OM and 2M males on all but a few of the 25 postnatal days of life. Also, 2M females were significantly heavier than OM females from Postnatal Day 6 onward, and 2M males weighed significantly more than OM males from Postnatal Day 19 onward. In a second study, food intake and body weight of animals from different intrauterine locations were examined from 25 to 120 days of age. Regardless of prior intrauterine position, males were always heavier than females. However, prior in utero location modulated body weight in that 2M females were significantly heavier than OM females and 2M males were reliably heavier than OM males. Intrauterine position effects were observed in the absence of any appreciable influence of this variable on levels of food intake. Taken together, the results suggest that prior in utero location may influence metabolic set points involved in the regulation of body weight and fat storage.     

Effect of corticosteroids on arachidonate induced mortality in male and female mice

Sodium arachidonate (50 mg/kg) given intravenously to male and female mice induces pulmonary emboli followed by respiratory distress and cyanosis. Female mice are significantly more resistant to this treatment than male mice. Cortisone pretreatment for four days to intact mice (10 mg/kg/day/4 days) had a significant protective effect in both males and females against arachidonate toxicity, eliminating the sex difference previously observed. Adrenalectomy four days before arachidonate infusion increased the sensitivity to SA and resulted in 100% mortality in both sexes. Pretreatment of adrenalized animals with cortisone significantly reduced to some degree the mortality rate in both sexes. Castration of male and female mice three weeks before adrenalectomy did not affect the mortality rate seen following adrenalectomy alone. In conclusion, exogenous cortical steroids augment the resistance of even intact mice and are absolutely necessary for survival in adrenalectomized animals. The observed sex differences in untreated intact animals is not seen after treatment with cortisone or adrenalectomy.     

Salivary epidermal growth factor and intestinal adaptation in male and female mice

Salivary epidermal growth factor (sEGF) levels are increased in male mice after small bowel resection (SBR) and may be important during intestinal adaptation. Since males have greater sEGF than females, the influence of sex on postresection adaptation was tested. Females had lower sEGF; however, sEGF substantially increased in both sexes after a massive (50%) SBR. Adaptive increases in DNA and protein content, villus height, and crypt depth, as well as crypt cell proliferation rates in the remnant ileum, were not different between males and females. Although significant postresection increases in sEGF were identified, EGF mRNA and protein did not change within the submandibular gland. Glandular kallikrein-13 and ileal EGF receptor expression were greater after SBR in female mice. Intestinal adaptation is equivalent in female and male mice after SBR. Despite lower sEGF, females demonstrated increased expression of a kallikrein responsible for sEGF precursor cleavage as well as amplified ileal EGF receptor expression. These results endorse an important differential response between sexes regarding sEGF mobilization and intestinal receptor availability during adaptation.