bluuebunny/crossref_metadata_2025 huggingface dataset:
A proton NMR study of the effect of a new intravasal injectable male contraceptive RISUG on seminal plasma metabolites
Authors: Sharma U , Chaudhury K , Jagannathan NR , Guha SK
Published: March 2004

Nuclear magnetic resonance (NMR) spectroscopy was used to quantify citrate, glucose, lactate, glycerophosphorylcholine and choline in seminal plasma from subjects injected with a new male contraceptive RISUG, a copolymer of styrene maleic anhydride dissolved in dimethyl sulphoxide, and in seminal plasma from normal ejaculates. No significant difference in the concentration of citrate was observed between the groups, indicating that the prostate is not affected by the contraceptive. The concentrations of glucose, lactate, glycerophosphorylcholine and choline were significantly lower (P < 0.01) in subjects injected with RISUG compared with controls. In addition, metabolite ratios such as choline:citrate, citrate:lactate, choline:lactate and glycerophosphorylcholine:choline were calculated. Citrate:lactate and glycerophosphorylcholine:choline ratios were significantly lower in RISUG-injected subjects than in controls (P < 0.01), thereby indicating the occurrence of partial obstructive azoospermia. The most important finding of the present study was that the intervention of RISUG in the vas deferens even for a period as long as 8 years is absolutely safe and does not lead to prostatic diseases.
Maternal nutrition and reproduction of daughters in wild house mice (Mus musculus)
Authors: Meikle D , Westberg M
Published: March 2004

Food deprivation after weaning often has greater effects on the reproductive success of females than of males. However, if animals are deprived prenatally (that is, through food deprivation of the mother during gestation), the reproductive success of males may be more adversely affected than that of females because of a disruption in the organizational effects of testosterone in neonatal male mice. The hypotheses that daughters of female mice deprived of food during gestation would have lower reproductive success than control daughters, but that the impact of maternal food deprivation would be lower for daughters than it would be for sons, was tested. There was no difference in the proportion of daughters of food-deprived and control mothers that produced one or two litters. However, the mean number of pups weaned in the second litters by daughters of control females (5.9 +/- 0.57 SEM) was greater than the number of pups weaned by daughters of food-deprived females (4.5 +/- 0.65 SEM). There were no differences in the mean birth or weaning body weights of offspring. Therefore, maternal food deprivation in mice may have a small but significant effect on the reproductive success of daughters. However, studies of sons born to females that were subjected to the same food deprivation protocol indicate that maternal food deprivation may have a much greater effect on the reproduction of sons than on that of daughters.
Transmission electron microscopy studies of the zona reaction in pig oocytes fertilized in vivo and in vitro
Authors: Funahashi H , Ekwall H , Kikuchi K , Rodriguez-Martinez H
Published: March 2004

The aim of this study was to determine the ultrastructure of cross-sectioned zonae pellucidae of in vitro-matured and ovulated pig oocytes before or after sperm penetration in vitro and in vivo, respectively. The in vitro and in vivo (ovulated) oocytes and zygotes (fertilized in vitro and in vivo) were fixed with glutaraldehyde either directly or after pretreatment with ruthenium red and saponin, processed and then examined using transmission electron microscopy. The thickness of the zona pellucida, as measured on the section of the specimens with largest diameter fixed with glutaraldehyde, differed between the in vivo (9.19 +/- 0.47 microm) and in vitro (5.95 +/- 0.51 microm) oocytes. The in vivo oocytes had a rather thick external mesh-like structure, whereas it was much thinner in the in vitro oocytes. This mesh-like external rim was less apparent in both in vivo and in vitro zygotes. Obvious differences in the density of the lattice formed by the fixed zonae pellucidae were visible between the outer and inner (ad-oolemmal) zonae. The outer area always formed a concentrically arrayed fibrillar network, whereas the inner area showed a much more compact, trabecule-like mesh. However, both areas, but particularly the outer network, were much more compacted after the zona reaction. Clear differences in the degree of fibrillar aggregation of the inner zona area were also observed between in vitro and in vivo zygotes, being much higher in the latter. This fibrillar network was more clearly visible in the zygotes pretreated with ruthenium red and saponin; the in vitro zygotes had a fibrillar, radially oriented set of parallel fibrils, whereas it was much more aggregated and trabecule-like in the in vivo zygotes. These results demonstrate that the fine structure of the zona pellucida and the zona reaction at sperm penetration differ between pig oocytes fertilized in vivo and in vitro. Moreover, the ultrastructure of the outer and inner pig zonae pellucidae has a different network organization.
Developmental consequences of embryo and cell manipulation in mice and farm animals
Authors: McEvoy TG , Robinson JJ , Sinclair KD
Published: March 2004

Advances in biotechnology in recent decades have revolutionized our understanding of early mammalian development and promise to provide ever more finely tuned and precisely targeted techniques for genetic enhancement of domestic animal species. In demonstrating what is both technically and biologically possible, not only in mice but also in larger animal species, research has provided hope that previously intractable diseases and genetic defects can be successfully combated. Crucial to this research is the ability to culture oocytes, embryos and somatic cells in vitro and to sustain their development without inducing adverse short- or long-term consequences. There is a need to refine current culture strategies in farm animal species to avoid jeopardizing their dependent technologies. A key to resolving current limitations of culture strategies is to identify, acknowledge and then address those features of in vitro culture that compromise early regulation of mammalian development. The aim of this review is to appraise critically in vitro embryo and somatic cell production strategies in the context of their impact on developmental competence and normality at embryonic, fetal and later stages. In addition, effects of physically manipulating embryos and cells, most notably via nuclear and gene transfer technologies, are considered with a view to identifying how detrimental consequences can be avoided.
Micronutrient programming of development throughout gestation
Authors: Ashworth CJ , Antipatis C
Published: March 2004

Vitamins and minerals serve essential roles in cellular metabolism, maintenance and growth throughout life. They are also central components of many enzymes and transcription factors. However, the need for optimum amounts of key micronutrients at critical stages during the periovulatory period and subsequent embryonic and fetal life has become the focus of sustained research activity only recently. In addition to folic acid, the minerals zinc, iron and copper and the antioxidant vitamins A and E are of particular importance during pregnancy. Both excesses and deficiencies of these micronutrients can have profound and sometimes persistent effects on many fetal tissues and organs in the absence of clinical signs of deficiency in the mother. The consequences of micronutrient imbalance on the developing conceptus may not be apparent at the time of the nutritional insult, but may be manifest later in development. However, supplementary micronutrients provided later in gestation or during postnatal life cannot completely reverse the detrimental effects of earlier micronutrient imbalance. Importantly, deficiency of a specific micronutrient, such as zinc, during pregnancy can result in a greater incidence of fetal malformation and resorptions than general undernutrition. Given the range of micronutrients that affect development, the number of developmental stages susceptible to inappropriate micronutrient status and the diverse biochemical systems and types of tissue affected, it is challenging to propose a unifying hypothesis that could explain the effects of micronutrient imbalance on programming throughout gestation. Micronutrient imbalance can affect pregnancy outcome through alterations in maternal and conceptus metabolism, as a consequence of their essential role in enzymes and transcription factors and through their involvement in signal transduction pathways that regulate development. Micronutrient-induced disturbances in the balance between the generation of free oxygen radicals and the production of antioxidants that scavenge free radicals may provide an additional mechanistic explanation. The detrimental effects of many micronutrient deficiencies, particularly zinc and copper, can be alleviated by supplementary antioxidants, whereas deficiencies of antioxidant vitamins A and E are likely to reduce defence against free radical damage.
Growth hormone and fertility in oMt1a-oGH transgenic mice
Authors: Thomas AD , Murray JD , Famula TR , Oberbauer AM
Published: March 2004

Female mice carrying a regulatable growth hormone transgene (oMt1a-oGH) are subfertile when the transgene is actively expressed. This study was designed to characterize subfertility caused by increased concentrations of growth hormone. In particular, this study aimed to: (i) determine the effects of transgene activation and inactivation on mating, conception, maintenance of pregnancy, ovulation rate, litter characteristics and embryonic survival at day 17 of pregnancy, (ii) characterize oestrous cyclicity in transgenic versus wild-type female mice, and (iii) correlate corticosterone concentrations with transgene expression and reproductive performance. Transgenic and wild-type female mice were allocated randomly to one of four treatment groups at weaning: (i) transgenic female mice that always express the transgene, (ii) transgenic female mice that never express the transgene, (iii) transgenic female mice that express the transgene for up to 8 weeks of age and (iv) non-transgenic wild-type female mice receiving the transgene stimulus until 8 weeks of age. Activation followed by inactivation of the transgene resulted in an increased incidence of remating, resulting in an extended interval to establish pregnancy in comparison with all other treatment groups. Transgenic mice that always expressed the transgene and those that expressed the transgene for up to 8 weeks of age had lower pregnancy rates and higher ovulation rates compared with mice from other treatment groups. Both embryonic survival and the duration of the oestrous cycle did not differ among treatment groups. Active expression of the transgene resulted in an increase in the plasma concentration of corticosterone, which was associated with reduced fertility. These data indicate that the presence of a high growth hormone concentration impedes the establishment and maintenance of pregnancy. Increased plasma corticosterone concentrations may interfere with implantation as well as potentiate leptin resistance, which has been reported previously in studies with these mice.
Hormonal characterization of the reproductive cycle and pregnancy in the female Mohor gazelle (Gazella dama mhorr)
Authors: Pickard AR , Abaigar T , Green DI , Holt WV , Cano M
Published: March 2004

The oestrous cycles of seven captive Mohor Gazelles (Gazella dama mhorr) were investigated. Hormone profiles obtained from faecal samples collected each day from cyclic females indicated that the mean duration of the oestrous cycle was 18.62 +/- 0.26 days (range 16-22 days; n = 37 oestrous cycles). No inter-individual differences in the concentration of faecal progestagen metabolites excreted were observed, but mean faecal oestrogen excretion during both the luteal and inter-luteal phases of the oestrous cycle varied among females (P < 0.001 and P = 0.070, respectively). Oestrous cycles were synchronized using controlled internal drug release (CIDR) devices, before natural mating with an intact male. Concentrations of faecal progestagen metabolites remained approximately constant for the first 10 weeks of gestation (mean +/- SEM = 4048 +/- 407 ng g(-1) faeces), before increasing to a mean of 23 556 +/- 1176 ng g(-1) faeces. Two of seven female gazelles conceived immediately after removal of the CIDR device, a similar proportion to that conceived at the postpartum oestrus under natural conditions. Life history data for these individuals indicated that the mean time to conception in female gazelles is positively correlated with peak values in the ratio of excreted oestrogen : progestagen during the inter-luteal period of their oestrous cycles (R(2) = 0.58; P < 0.05). This finding indicates that interactions between steroid production and metabolism may influence the likelihood of conception occurring in this species.
An in situ hybridization study of the effects of artificial insemination on the localization of cells expressing MHC class II mRNA in the chicken oviduct
Authors: Zheng WM , Nishibori M , Isobe N , Yoshimura Y
Published: March 2004

The aim of this study was to determine the effects of artificial insemination on the localization of antigen-presenting cells expressing MHC class II mRNA in chicken oviducts. Laying hens (35 weeks old) were inseminated with fresh semen or sham-inseminated with saline daily for 3 days. In situ hybridization was performed to detect chicken MHC class II (B-LB21 major gene) mRNA on frozen sections of oviductal infundibulum, uterovaginal junction and vagina by using digoxigenin-labelled PCR probes. Cells expressing MHC class II were observed mainly in the oviductal mucosal stroma and occasionally in the mucosal epithelium. After 24 h, the population of cells expressing MHC class II in the infundibulum was significantly higher in laying hens inseminated with fresh semen than in the control hens sham-inseminated with saline (P < 0.05). However, there was no significant difference in the population of cells expressing MHC class II in the uterovaginal junction and vagina between the artificially inseminated and control hens. These results indicate that anti-sperm immune responses, including the influx of cells expressing MHC class II and enhanced MHC class II mRNA expression, probably occur in the infundibulum after artificial insemination.
Benefit of FSH priming of women with PCOS to the in vitro maturation procedure and the outcome: a randomized prospective study
Authors: Mikkelsen AL , Lindenberg S
Published: March 2004

The aim of this study was to determine whether the rates of in vitro oocyte maturation, fertilization and cleavage, as well as implantation rate and pregnancy rate, could be improved by low-dose priming with FSH in vivo before retrieval of immature oocytes in patients with polycystic ovary syndrome (PCOS). From March 1998 to June 2000, a total of 28 women underwent 36 completed treatment cycles, randomized sequentially in one of two groups. Women in group 1 (n = 12 cycles) received no stimulation and women in group 2 (n = 24 cycles) received 150 iu recombinant FSH day(-1) for 3 days, initiated on day 3 after menstruation. Aspiration was performed transvaginally between day 9 and day 17 in the unstimulated group and on day 8 or day 9 in the FSH-primed group after FSH deprivation for 2 or 3 days. All cumulus-enclosed oocytes of healthy appearance were matured in culture medium (TCM-199) in vitro for 28-36 h before intracytoplasmic sperm injection (ICSI). After oocyte retrieval the women were given oestradiol (6 mg day(-1)) and progesterone administration (300 mg day(-1)) was initiated 2 days later. Suitable embryos (maximum two embryos) were transferred on day 3 after ICSI. The percentage of oocytes reaching metaphase II was significantly higher (P < 0.05) in the FSH-primed group (59%, 92/156) compared with the non-primed group (44%, 36/81). There were no significant differences in the rates of oocyte fertilization and cleavage between these groups. No pregnancies were obtained in group 1 (0%, 0/12), whereas seven clinical pregnancies were obtained in group 2 (29%, 7/24) (P < 0.05). In group 2, 37 embryo transfers resulted in eight implantations (21.6%). Three healthy singleton children have been born at term; the remaining pregnancies ended with spontaneous abortions in the first trimester. These results indicate that priming with recombinant FSH before harvesting of immature oocytes from patients with PCOS may improve the maturational potential of the oocytes and the implantation rate of the cleaved embryos.
Successful production of offspring after superovulation and in vitro culture of embryos from domestic ferrets (Mustela putorius furos)
Authors: Li ZY , Jiang QS , Zhang YL , Liu XM , Engelhardt JF
Published: March 2004

In an effort to expand the use of ferrets as models for genetic disease, several experimental parameters that are required for successful genetic manipulation in this species were investigated. Optimum superovulation (19.3 +/- 0.6 oocytes and embryos per female) was achieved after injections of 100 iu equine chorionic gonadotrophin (eCG) and 150 iu human chorionic gonadotrophin (hCG). The ovulation rate achieved by the treatment was more than double that induced by mating. Mating with a male immediately after hCG treatment did not significantly alter the number of oocytes ovulated or the number of embryos present, indicating that mating is not required for superovulation in ferrets. Of embryos harvested at the one-cell stage, 64.5% and 47.1% developed into blastocysts when cultured in vitro in CZB or TCM-199 plus 10% fetal bovine serum (FBS) media, respectively. In contrast, only 17.1% of embryos cultured in vitro in NCSU-23 developed to the blastocyst stage. Both freshly retrieved and in vitro cultured embryos from cinnamon-coloured parents produced live young when transferred at the eight-cell stage into albino, pseudo-pregnant recipients. The percentage of kits delivered relative to embryos transferred was 61% for freshly retrieved embryos and 32% for embryos cultured in vitro. These results demonstrate successful embryo transfer in ferrets and provide a basis for further study of genetic modelling approaches in this species after embryo manipulation.
Involvement of connexin 43 in meiotic maturation of bovine oocytes
Authors: Vozzi C , Formenton A , Chanson A , Senn A , Sahli R , Shaw P , Nicod P , Germond M , Haefliger JA
Published: March 2004

In ovarian follicles, cumulus cells provide the oocyte with small molecules that permit growth and control maturation. These nutrients reach the germinal cell through gap junction channels, which are present between the cumulus cells and the oocyte, and between the cumulus cells. In this study the involvement of intercellular communication mediated by gap junction channels on oocyte maturation of in vitro cultured bovine cumulus-oocyte complexes (COCs) was investigated. The stages of oocyte maturation were determined by Hoechst 33342 staining, which showed that 90% of COCs placed in the maturation medium for 24 h progress to the metaphase II stage. Bovine COC gap junction communication was disrupted initially using n-alkanols, which inhibit any passage through gap junctions. In the presence of 1-heptanol (3 mmol l(-1)) or octanol (3.0 mmol l(-1) and 0.3 mmol l(-1)), only 29% of the COCs reached metaphase II. Removal of the uncoupling agent was associated with restoration of oocyte maturation, indicating that treatment with n-alkanols was neither cytotoxic nor irreversible. Concentrations of connexin 43 (Cx43), the major gap junction protein expressed in the COCs, were decreased specifically using a recombinant adenovirus expressing the antisense Cx43 cDNA (Ad-asCx43). The efficacy of adenoviral infection was > 95% in cumulus cells evaluated after infection with recombinant adenoviruses expressing the green fluorescence protein. RT-PCR performed on total RNA isolated from Ad-asCx43-infected COCs showed that the rat Cx43 cDNA was transcribed. Western blot analysis revealed a three-fold decrease in Cx43 expression in COCs expressing the antisense RNA for Cx43. Injection of cumulus cells with Lucifer yellow demonstrated further that the resulting lower amount of Cx43 in infected COCs is associated with a two-fold decrease in the extent of coupling between cumulus cells. In addition, oocyte maturation was decreased by 50% in the infected COC cultures. These results indicate that Cx43-mediated communication between cumulus cells plays a crucial role in maturation of bovine oocytes.
Pale vacuolated epithelial cells in epididymis of aflatoxin-treated mice
Authors: Agnes VF , Akbarsha MA
Published: March 2004

The responses of the mouse epididymal epithelium to subchronic doses of aflatoxin B1 were investigated in a histological study. Either few and large or small and profuse vacuoles containing an amorphous to dense periodic acid-Schiff-positive material were observed in the epithelium of all the segments of the epididymis. Resin-embedded semi-thin sections and transmission electron microscopy indicated that these vacuoles were intracellular. The cells that contained these vacuoles were quite different in organization and electron density from the cell types already established in the epididymal epithelium and are designated as pale vacuolated epithelial cells. Owing to aflatoxin B1 toxicity, the apical membrane of some of the principal cells, either individually or in groups, disintegrated so that the principal cells released their contents into the lumen of the duct through development of a 'fistula'. Spermatozoa from the ductal lumen entered the principal cell fistula and reached the basal lamina. If extravasation of the spermatozoa via this route occurred, it would bring about an autoimmune response, leading to the formation of spermatic granulomas and the generation of anti-sperm antibodies. Extravasation of spermatozoa seems to be offset by the underlying basal cell, which is presumed to develop into a pale vacuolated epithelial cell to enclose the disintegrating principal cells and the spermatozoa arriving at the principal cell. Thus, the development of pale vacuolated epithelial cells may be a protective device preventing an autoimmune response to sperm antigens in the context of toxicant-induced degeneration of the principal cells of the epididymal epithelium.
Inhibition of mouse fertilization in vivo by intra-oviductal injection of an anti-equatorin monoclonal antibody
Authors: Yoshinaga K , Saxena DK , Oh-oka T , Tanii I , Toshimori K
Published: March 2004

The monoclonal antibody mMN9 recognizes an antigenic molecule, equatorin, which is localized at the equatorial segment of the mammalian sperm acrosome. Our previous results using an IVF system indicated that mMN9 blocked sperm-oocyte fusion. Antibody-containing and control solutions were injected directly into the right and left oviductal ampullae, respectively, of anaesthetized female mice to assess the effect of mMN9 on fertilization in vivo. After hCG treatment, the females were mated, and their oviductal eggs and implanted embryos were examined. mMN9 was retained in the oviductal lumen at 20 h after injection. The rates of fertilization and concomitant pregnancy were significantly lower than in the control side (P < 0.05). In addition, histological studies showed no evidence of pathological changes in the female reproductive tract after the injections. These results indicate that mMN9 inhibits mouse fertilization significantly under in vivo conditions and that this injection method should be useful for studying the effects of antibodies and agents on fertilization in vivo.
Measurement of apoptosis in long-term cultures of human ovarian tissue
Authors: Rahimi G , Isachenko E , Sauer H , Wartenberg M , Isachenko V , Hescheler J , Mallmann P , Nawroth F
Published: March 2004

At present, the long-term culture of ovarian tissue is problematic. The aim of this study was to measure apoptosis in long-term cultures of human ovarian tissue. Biopsies of human ovaries were cultured for 6 weeks. Samples were taken weekly for histological investigation. The apoptotic cells were marked with anti-caspase 3. Simultaneous to this experiment, other tissue samples were preincubated for 3 h with 1 micromol staurosporine l(-1), an inducer of apoptosis, and apoptosis was compared among samples. Furthermore, the proportion of lethal cells was determined weekly. After 6 weeks, 99% of the tissue samples showed an intact structure. They expanded in all directions on the floor of the multi-wells to form a monolayer. Apoptotic cells could be marked only sporadically (16.3 +/- 5.9 fluorescence (counts per 3600 microm(2))) after 6 weeks. After preincubation with staurosporine after the same period of culture, the proportion of apoptotic cells was significantly increased compared with that in untreated control samples (66.8 +/- 14.5 versus 16.3 +/- 5.9%, respectively; P < 0.05). Under the same experimental conditions, the proportion of lethal cells was 3.6 +/- 0.9, 3.9 +/- 2.1 and 5.2 +/- 1.5% for weeks 1, 3 and 6, respectively. After preincubation with 1 micromol staurosporine l(-1), the proportion of pyknotic cells after 6 weeks of culture was significantly higher (37.2 +/- 4.4%) than that in control samples (3.95 +/- 2.05%; P < 0.05). No significant increase in apoptosis in cultured human ovarian tissue after 6 weeks was observed compared with control tissues on day 1. These results indicate that under optimal culture conditions it is possible to cultivate human ovarian tissue long term. The influence of long-term culture on hormone synthesis and follicle maturity will be investigated further.
Immune cells in the corpus luteum: friends or foes?
Authors: Pate JL , Landis Keyes P
Published: March 2004

The corpus luteum produces progesterone, which is essential for the maintenance of pregnancy. In the absence of a viable embryo, the corpus luteum must regress rapidly to allow for development of new ovulatory follicles. In many species, luteal regression is initiated by uterine release of PGF(2alpha), which inhibits steroidogenesis and may launch a cascade of events leading to the ultimate demise of the tissue. Immune cells, primarily macrophages and T lymphocytes, are present in the corpus luteum, particularly at the time of luteolysis. The macrophages are important for ingestion of cellular remnants that result from the death of luteal cells. However, it has also been hypothesized that immune cells are involved directly in the destruction of luteal cells, as well as in the loss of steroidogenesis; this hypothesis is reviewed in the first part of this article. An alternative hypothesis is also presented, namely that immune cells serve to abate an inflammatory response generated by dead and dying luteal cells, in effect, preventing a response that would otherwise damage surrounding ovarian tissues. Finally, the changes in immune cells that accompany maternal recognition of pregnancy and rescue of the corpus luteum are discussed briefly. Inhibition of immune cells in the corpus luteum during early pregnancy may be due to embryonic or uterine signals, or to maintenance of high progesterone concentrations within the luteal tissue.
Mediation of seasonal testicular regression by apoptosis
Authors: Young KA , Nelson RJ
Published: March 2004

Animals restrict breeding to specific times of the year when reproductive success is most likely. In males, the most reliable method of reproductive inhibition is gonadal retrogression to an immature state. Depending on the species, the testes decrease in size by between 10 and 95% in response to environmental cues associated with the non-breeding season. An increased rate of apoptosis can occur during testicular regression, whereas little testicular apoptosis is observed during testicular recrudescence or during the breeding season. The Fas system is implicated as a potential mechanism to transmit the apoptotic signal to germ cells during regression, although many apoptotic pathways probably contribute to testicular regression. Finally, differences in both the contribution of testosterone and in the types of cell that undergo apoptosis differ between rodents and birds, indicating that different cellular mechanisms and possibly signals have evolved at the testis to suspend reproduction on a seasonal basis.
Characteristics of infertility in female hypothyroid (hyt) mice
Authors: Jiang JY , Imai Y , Umezu M , Sato E
Published: March 2004

Female hypothyroid (hyt) mice are infertile, but the reason for this infertility is not yet known. The present study was conducted to determine whether hypothyroidism induced infertility in immature and mature hyt mice. Furthermore, animals were treated with thyroxine and gonadotrophins at different times to determine whether infertility was due to failure of follicular development, implantation or pregnancy. There were no significant differences in the numbers of ovulated eggs induced by gonadotrophin treatment or the percentages of eggs developed in vitro among immature normal controls, hyt and thyroxine-treated hyt mice. Mature hyt mice showed continuous dioestrus, and ovulated significantly fewer eggs after gonadotrophin treatment and failed to establish pregnancy after mating compared with mature control mice. Mature hyt mice had significantly fewer corpora lutea > 500 microm in diameter and significantly lower progesterone concentrations. Thyroxine treatment before mating in hyt mice resulted in well-developed corpora lutea, an increase in progesterone and normal pregnancy, regardless of subsequent thyroxine administration. In conclusion, infertility occurs in mature rather than immature hyt mice, is due to the failure of follicular development and pregnancy, and can be reversed by thyroxine treatment before mating.
Autoradiographic determination of angiotensin II receptors in prepubertal and postpubertal pig ovarian tissue
Authors: Shuttleworth G , Hunter MG , Broughton Pipkin F
Published: March 2004

The aim of this study was to determine whether specific receptors for angiotensin II are present in prepubertal and postpubertal pig ovaries using an autoradiographic technique and computerized densitometry. Frozen sections were cut from prepared samples, and (125)I-labelled angiotensin II and the angiotensin II receptor subtype-specific nonpeptide antagonists for receptor subtype 1, AT(1) (GR117289) and subtype 2, AT(2) (PD123319) were used. In both pre- and postpubertal pig ovarian tissue, specific receptors for angiotensin II were demonstrated. These receptors had a density of 2487.6 (range: 267.5-5177.6, n = 4) and 3703.8 (range: 1819.9-5207.8, n = 4) fmol per mm(2), respectively, and dissociation constants of 130.0 and 26.3 nmol l(-1), respectively (prepubertal ovarian range: 106.0-165.4 nmol l(-1); postpubertal ovarian range: 26.1-100.3 nmol l(-1); P < 0.05, Mann-Whitney U test). AT(1) receptors with a K(i) for (125)I-labelled angiotensin II of 346.9 nmol l(-1) in the prepubertal and 268.1 nmol l(-1) in the postpubertal ovary were located predominantly in follicle wall tissue. Competitive inhibition studies using both angiotensin II antagonists resulted in a decrease in K(i) with prepubertal tissue (283.7 nmol l(-1)) and an increase in postpubertal tissue (293.9 nmol l(-1)). Immunocytochemistry using sections from paraffin wax-embedded prepubertal (n = 4) and postpubertal (n = 4) pig ovaries confirmed the presence of AT(1) receptors on the granulosa cell layer, but not the thecal cell layer, of antral follicles in both pre- and postpubertal pig ovarian tissue, and AT(2) receptors within the granulosa cell layer of prepubertal pig ovarian antral follicles. In summary, these results indicate that angiotensin II receptors are of higher affinity in postpubertal tissue than they are in prepubertal tissue, and indicate an active renin-angiotensin system within the pig ovary.
Effect of acute administration of cocaine on pituitary gonadotrophin secretion in female rats
Authors: King TS , McNichol M , Canez MS , Javors MA , Schenken RS
Published: March 2004

The aim of this study was to characterize the acute effects of cocaine administration on pituitary gonadotrophin secretion in adult female rats. Ovariectomized, oestradiol-treated rats were infused i.v. with 0.1 ml normal saline or 2 mg cocaine hydrochloride kg(-1). Blood samples were collected immediately before cocaine infusion and at 3, 10, 30 and 60 min after cocaine infusion. Circulating LH concentrations were increased by 10 min after cocaine administration (P < 0.05 versus saline-treated controls) and decreased thereafter. Serum FSH concentrations were not significantly different from those of saline-infused controls at any time. In a second experiment, oestradiol-treated, ovariectomized rats were infused i.v. with: saline only, 2 mg cocaine hydrochloride kg(-1) in saline, 200 ng synthetic GnRH in 100 microl PBS or 200 ng synthetic GnRH plus 2 mg cocaine hydrochloride kg(-1) in PBS. Blood samples were collected immediately before drug infusions and 20 min later. Cocaine had no effect on either GnRH-stimulated LH or FSH secretion. In a third experiment, pituitary cells were obtained from oestradiol-treated, ovariectomized rats. The cell cultures were exposed to 25 ng cocaine hydrochloride ml(-1), 10(-10)-10(-7) mol GnRH (-1) with and without 25 ng cocaine hydrochloride ml(-1), or vehicle only. Medium was collected before and after exposure to GnRH to determine concentrations of secreted LH and FSH. Similar to the results of the second study, cocaine had no effect on GnRH-stimulated LH or FSH secretion from pituitary cells in vitro. On the basis of these results it is suggested that acutely administered cocaine stimulates release of hypothalamic GnRH, which, in turn, stimulates pituitary gonadotrophin secretion. Acute administration of cocaine does not appear to affect pituitary gonadotrophin secretion directly.
Distribution of vascular endothelial growth factor (VEGF) and its binding sites at the maternal-fetal interface during gestation in pigs
Authors: Charnock-Jones DS , Clark DE , Licence D , Day K , Wooding FB , Smith SK
Published: March 2004

Pigs show epitheliochorial placentation, in which the maternal uterine epithelium and the fetal trophectoderm become closely apposed. There is no invasion of trophoblast into the maternal tissue, and nutrient and waste exchange take place across two epithelial layers beneath which a complex network of capillaries forms. Later in gestation, the epithelial cells become indented by blood vessels, which greatly reduces the distance for diffusion between the two circulatory systems. Vascular endothelial growth factor is a secreted homodimeric angiogenic growth factor that is involved in physiological and pathological angiogenesis. Its receptors are generally restricted to endothelial cells. Ligand binding, in situ hybridization and immunohistochemistry were carried out in pig placenta throughout gestation to investigate the possible role of vascular endothelial growth factor and its receptors in non-invasive placentation. In situ hybridization and immunohistochemistry revealed that mRNA and immunoreactivity for vascular endothelial growth factor were localized in both maternal and fetal epithelial cells at the maternal-fetal interface and over the maternal glands, although the signal was generally weaker in the maternal glands. Ligand binding was used to localize for vascular endothelial growth factor receptors; no binding was observed over the maternal glands, but very strong binding was localized to the endometrial blood vessels. At the interface between maternal and fetal tissue, a similar pattern was observed whereby the numerous small capillaries at the bases of the two apposed epithelia bound vascular endothelial growth factor specifically. It is concluded that vascular endothelial growth factor produced by the maternal and fetal epithelial layers promotes the growth of capillaries locally, which would facilitate the development of two vascular networks for the efficient transfer of nutrients and waste products.
Induced ovulation, mating success and embryonic development in the stripe-faced dunnart, Sminthopsis macroura
Authors: Hickford DE , Merry NE , Johnson MH , Selwood L
Published: March 2004

Induced ovulation resulting in normal embryos is rare in marsupials. In this study natural and induced ovulations were compared in mature Sminthopsis macroura (n = 122). Comparison of maturation of preovulatory oocytes by ovarian histology and examination of oocytes removed from developing follicles in 12 ovaries of 23 animals receiving 0.058 iu equine serum gonadotrophin (eSG) g(-1) with ovaries of 12 animals undergoing natural cycles showed that oocyte maturation was significantly more irregular when it was induced (P < 0.001). Postovulatory stages were examined by estimating the number of eggs ovulated from ovarian histology, and by counting oviduct and uterine contents recovered after ovulation. S. macroura receiving 0.087 iu eSG g(-1) (n = 34), administered as one (n = 17) or two (n = 17) injections, were significantly (P < 0.05) more likely to ovulate (74%), mate (80%) and have conceptuses (66%) than were animals receiving 0.058 iu eSG g(-1) (12, 53 and 0%, respectively) (n = 17), and the values were similar to those in animals (n = 36) undergoing natural cycles (100, 81 and 56%, respectively). Induced ovulation using 0.087 iu eSG g(-1) yielded significantly (P < 0.05) more oocytes per ovary (20.8 +/- 8.5; combined data) than did ovulation in animals undergoing natural cycles (13.7 +/- 3.2) (ANOVA, t test). The responses of animals induced in different phases of the oestrous cycle with 0.087 iu eSG g(-1) were not significantly different (ANOVA) with respect to the number of corpora lutea per ovary, conceptuses per animal or days to ovulation after injection. However, the proportion of females that responded after receiving 0.058 iu eSG g(-1) in the luteal phase was significantly different from that in animals treated with the same dose in the intermediate phase (P < 0.01) and in non-cyclic females treated with 0.058 iu eSG g(-1) (P < 0.02). The main benefits of the treatment were that normal embryos resulted and that 70-78% of non-cyclic animals could be induced to ovulate.
Maternal nutritional manipulation of placental growth and glucose transporter 1 (GLUT-1) abundance in sheep
Authors: Dandrea J , Wilson V , Gopalakrishnan G , Heasman L , Budge H , Stephenson T , Symonds ME
Published: March 2004

Glucose transporter 1 (GLUT-1) is the predominant glucose transporter in the placenta but the extent to which its abundance is nutritionally regulated is unknown. This study investigated the effects of restricted maternal nutrition between day 28 and day 80 of gestation followed by re-feeding to either meet or to exceed the total energy requirements on placental size and GLUT-1 abundance at mid-gestation (that is, day 80) and near to term (that is, days 140-145 of gestation; term = 147 days). Singleton bearing ewes either consumed 8.7-9.9 MJ day(-1) of metabolizable energy (that is, well fed) or 3.2-3.8 MJ day(-1) of metabolizable energy (that is, nutrient restricted) from day 28 to day 80 of gestation, after which stage they consumed either 6.5-7.5 MJ day(-1) (that is, adequately fed) or 8.0-10.9 MJ day(-1) (that is, well fed) of metabolizable energy until near to term. In all ewes, at both sampling dates, the abundance of GLUT-1 was higher in the maternal component than in the fetal component of the placenta. Immunohistochemistry confirmed that GLUT-1 was located in the maternal uterine syncytium. At day 80 of gestation, placental mass was lower (P < 0.05) in the nutrient restricted group, but there was no difference in the abundance of GLUT-1 between the nutrient restricted group and the well fed group. At near term, placental mass was greater (P < 0.05) in ewes that were nutrient restricted during early to mid-gestation and then adequately fed up to term compared with ewes that were well fed during early to mid-gestation. This increase was associated with a higher (P < 0.05) abundance of total placental GLUT-1 and a larger fetus. There was no effect of previous nutrient restriction on placental mass, fetal weight or GLUT-1 abundance at term, when ewes were well fed in the second half of gestation. In conclusion, maternal nutrient restriction between early to mid-gestation alters placental growth but has no effect on placental GLUT-1 abundance. Increasing maternal feed intake to meet calculated energy requirements in previously nutrient restricted ewes during the second half of gestation, increases placental mass and fetal weight, and the abundance of GLUT-1, an adaptation not observed if maternal food intake is increased above requirements.
Refrigeration of donor cells in preparation for bovine somatic nuclear transfer
Authors: Liu JL , Wang MK , Sun QY , Zhang XR , Jiang LK , Chen DY
Published: March 2004

In mammals, preparation of donor cells for somatic nuclear transfer is very important because the character of the donor cell directly affects the efficiency and outcome of transfer. The protocols used most commonly for donor preparation are (i) disaggregating cells from fresh tissue 1-2 h before micromanipulation or (ii) trypsinizing cultured cells temporarily, after special treatments for 3-8 days (for example, serum starvation). In this study, a new simple protocol was designed, whereby the donor cells (cumulus cells) used in bovine somatic nuclear transfer were refrigerated. In brief, cultured cells at 80-100% confluency were detached using trypsin, washed by centrifugation, aliquoted into different vials and refrigerated at 4 degrees C. The density of viable cells was decreased after day 1 of refrigeration; however, the rate of decrease tended to slow down with increasing duration of refrigeration. Cells refrigerated for 15 days were seeded at a density of 5 x 10(4) ml(-1) and reached 70% confluency after day 2 of culture. Most cells had the normal number of chromosomes (2n = 60). Cells chilled at 4 degrees C for different durations were removed from refrigeration and immediately subjected to micromanipulation. The in vitro development of reconstructed embryos (fusion rates, cleavage rates, morula and blastocyst rates) indicated that there were no significant differences among treatment groups regardless of the duration of refrigeration (0-2 weeks) of the donor cells. Reconstructed embryos were transferred into the uteri of recipient cows. No significant differences were observed in established early pregnancies between embryos derived from the non-refrigerated donor cells and those derived from refrigerated donor cells. This study indicates that refrigeration of donor cells for 1-2 weeks is a feasible protocol for preparing donor cells for bovine somatic nuclear transfer, and does not compromise development in vitro and early development in vivo.
Superovulation, fertilization and in vitro embryo development in mice after administration of an inhibin-neutralizing antiserum
Authors: Wang H , Herath CB , Xia G , Watanabe G , Taya K
Published: March 2004

The present study was conducted to investigate the effect of immunoneutralization against endogenous inhibin on oocyte and embryo production in adult and immature mice. At 12:00 h on day 2 of oestrus (day 1 of dioestrus), a single i.p. injection of inhibin antiserum (50, 100, 200 or 400 microl per animal) or equine chorionic gonadotrophin (eCG; 10 or 20 iu per animal) or control goat serum (100 microl per animal) was administered to adult female mice. After 48 h, the mice in each of the three groups were given a single i.p. injection of hCG (10 iu per animal). At 42 h after hCG injection, ova were collected from oviducts and cultured in KSOM solution. Treatments with both inhibin antiserum-hCG and eCG-hCG induced superovulation in all the animals tested. The number of oocytes in animals treated with inhibin antiserum was significantly higher (P < 0.05) compared with the control group, and the number of oocytes ovulated in animals treated with 200 or 400 ml inhibin antiserum was significantly (P < 0.05) higher than that in animals treated with 10 or 20 iu eCG. The superovulated oocytes that were fertilized normally in vivo were able to form blastocysts in vitro. The rate of blastocyst development for animals treated with 50-200 ml inhibin antiserum was significantly (P < 0.05) higher than that of the eCG-treated animals. Irrespective of the day of the oestrous cycle, 200 microl inhibin antiserum administered at 12:00 h on each of 4 days induced superovulation in all the animals tested. The rates of oocyte and embryo production by these animals were significantly (P < 0.05) higher than in the control groups. Furthermore, administration of inhibin antiserum at doses of 50, 100, 200 or 400 ml produced similar results in 26-day-old immature mice. These results indicate that passive immunoneutralization of endogenous inhibin alpha-subunit induces superovulation in immature and adult mice. The superovulated oocytes obtained by administration of inhibin antiserum have normal embryonic developmental competence. Thus, it is concluded that this inhibin antiserum method is a new practical alternative for induction of superovulation in mice instead of the more commonly used eCG-hCG protocol.
Oocyte control of ovarian follicular development and function in mammals
Authors: Eppig JJ
Published: March 2004

A new perspective on ovarian follicular development has emerged over the last decade. Whereas the oocyte was previously considered only a passive recipient of developmental signals from oocyte-associated granulosa cells, it is now clear that communication between oocytes and granulosa cells is bidirectional. A complex interplay of regulatory factors governs the development of both types of cell. This interplay is essential not only for oocyte development but also for follicular development, beginning with the initial assembly of the primordial follicle and continuing throughout ovulation. The existence of an oocyte-granulosa cell regulatory loop, essential for normal follicular differentiation as well as for the production of an oocyte competent to undergo fertilization and embryogenesis, is proposed. Although gonadotrophins are essential for driving the differentiation of granulosa cell phenotypes, within its sphere of influence, the oocyte is probably the dominant factor determining the direction of differentiation and the function of the granulosa cells associated with it.
Potential role of a sperm-derived phospholipase C in triggering the egg-activating Ca2+ signal at fertilization
Authors: Swann K , Parrington J , Jones KT
Published: March 2004

An increase in intracellular Ca2+ at fertilization is the trigger for egg activation in all species that have been studied. Exactly how sperm-egg interaction leads to this Ca2+ increase has not been established. There is increasing support for the hypothesis that the spermatozoon introduces a Ca2+-releasing protein into the egg cytoplasm after gamete membrane fusion. This review discusses the merits of this 'sperm factor' hypothesis and presents evidence indicating that the sperm factor, at least in mammals, consists of a phospholipase C with distinctive properties. This evidence leads us to propose that, after gamete fusion, a sperm-derived phospholipase C causes production of inositol 1,4,5- trisphosphate, which then generates Ca2+ waves from within the egg cytoplasm.