bluuebunny/crossref_metadata_2025 huggingface dataset:
Oestrogenic effects of neonatal administration of raloxifene on hypothalamic-pituitary-gonadal axis in male and female rats
Authors: Pinilla L , Gonzalez LC , Gaytan F , Tena-Sempere M , Aguilar E
Published: March 2004

Selective oestrogen receptor modulators constitute a family of drugs that are used increasingly in the management of oestrogen-associated pathology. Raloxifene is a selective oestrogen receptor modulator that is used to treat and prevent osteoporosis in post-menopausal women. The actions of raloxifene on bone, breast, uterus and serum cholesterol concentrations have been widely analysed, but very few studies have investigated the possible actions of this drug on the central nervous system. The central nervous system of the newborn rat is very sensitive to oestrogen action. In this study a series of experiments was conducted to analyse the effects of different doses of raloxifene (50, 100, 250 or 500 microg per rat per day) administered to neonatal rats on days 1-5 of age. Female rats treated with raloxifene showed decreased gonadotrophin secretion, hyperprolactinaemia, advanced vaginal opening, decreased body weight, persistent presence of cornified epithelial cells in vaginal smears, anovulation, inhibition of positive feedback between oestradiol and LH, and infertility. Male rats showed delayed balanopreputial separation, reduced body weight and hyperprolactinaemia. All these changes resemble those obtained after neonatal administration of oestradiol benzoate, thus indicating, for the first time, that raloxifene exerts an oestrogenic action on the hypothalamic-pituitary structures controlling reproductive function in rats.
Significance of serum early pregnancy factor concentrations during pregnancy and embryonic development in Sminthopsis macroura (Spencer) (Marsupialia: Dasyuridae)
Authors: Cruz YP , Selwood L , Morton H , Cavanagh AC
Published: March 2004

Marsupial pregnancy differs from that in eutherians in duration, placentation and hormonal profile so much so that maternal recognition of pregnancy may not occur in polyovular marsupials. However, a comparison of gravid and non-gravid uteri reveals differences indicative of histological and physiological adaptations to pregnancy. In the present study, the hypothesis that embryo-maternal signalling occurs in polyovular marsupials was tested by examining serum from non-pregnant and pregnant Sminthopsis macroura for the presence of early pregnancy factor (EPF), a serum protein secreted by the ovary in response to the presence of a newly fertilized egg in the oviduct. EPF is detectable in the serum of pregnant, but not in non-pregnant, females in all eutherians studied to date. In the present study, EPF was detected in S. macroura serum by the rosette inhibition test during the first 9 days of the 10.7 day gestation period in this marsupial. However, EPF was not detected on day 10, just before parturition, or in non-pregnant or preovulatory animals. Immunohistochemical analysis of ovaries from gravid and non-gravid animals demonstrates that EPF is found in the capillaries, interstitial spaces and secretory cells of the corpus luteum. It is concluded that the spatiotemporal pattern of EPF activity described strongly indicates that maternal recognition of pregnancy in marsupials is mediated, at least in part, by EPF. Because the endocrinological milieu is the same in pregnant and non-pregnant marsupials, the possibility of using marsupials as an experimental system for studying EPF function unconfounded by hormonal effects is presented.
Changes in the localization of MHC class II positive cells in hen ovarian follicles during the processes of follicular growth, postovulatory regression and atresia
Authors: Barua A , Michiue H , Yoshimura Y
Published: March 2004

The aim of this study was to determine the changes in the population of major histocompatibility complex class II positive (MHC-II(+)) cells in ovarian follicles during the processes of follicular growth, postovulatory regression and follicular atresia in hens. Cryostat sections of ovarian stroma containing cortical follicles, small white follicles, the largest (F(1)) and third largest (F(3)) preovulatory follicles, postovulatory and atretic follicles of laying hens were prepared. The sections were immunostained for MHC-II molecules using mouse anti-chicken MHC-II monoclonal antibody and positive cells were counted using a computer-assisted image analyser under a light microscope. MHC-II(+) cells were localized in the theca layer of normally growing follicles including cortical follicles, small white follicles and F(3) and F(1) preovulatory follicles, whereas they were found in both the theca and granulosa layers in postovulatory and atretic follicles. The frequency of MHC-II(+) cells in the theca layer was significantly increased during follicular growth from cortical follicles to F(3) preovulatory follicles. Although the population of MHC-II(+) cells did not differ between F(3) and F(1) preovulatory follicles, it increased significantly in postovulatory follicles (P < 0.01). The population of MHC-II(+) cells was significantly greater in the theca layer of atretic follicles than in non-atretic follicles (P < 0.01). These results indicate that the antigen-presenting function via MHC-II increases in association with follicular growth. A marked increase in MHC-II(+) cells indicates that these cells may be involved in regression of postovulatory and atretic follicular tissues.
Comparative aspects of conceptus growth: a historical perspective
Authors: Betteridge KJ
Published: March 2004

This review provides a historical background for a series of articles arising from a symposium on Early Regulation of Mammalian Development. It begins by tracing connections between the work of J. Cossar Ewart and the first cloning of mammals by somatic cell nuclear transfer. Historical scientific landmarks over the last century that have affected our attitudes to 'fetal autonomy' and conceptus development are reviewed briefly, as are salient studies that have established the importance of nutrition to pre- and postnatal life. Finally, attention is drawn to some differences in conceptus development among various mammals, and the argument made that understanding these differences might be of benefit to the development of reproductive technology.
Effect of cytokinesis inhibitors, DMSO and the timing of oocyte activation on mouse cloning using cumulus cell nuclei
Authors: Wakayama T , Yanagimachi R
Published: March 2004

Cloning methods are now well described and in almost routine use. However, the frequencies of production of live offspring from activated oocytes remain at < 3% and little is known about the factors that affect these frequencies. The effects of cytokinesis inhibitors, dimethylsulphoxide (DMSO) and the cell cycle of recipient cytoplasm on the cloning of mice were examined. Reconstructed oocytes, which were activated immediately after nucleus injection and cultured without cytochalasin B, developed into blastocysts at a frequency of 30--54% and into live cloned offspring at a frequency of 2--3%. Activated zygotes did not support development to full term after nuclear transfer. Reconstructed oocytes were activated 1--3 h after nuclear transfer and were exposed separately to three inhibitors of cytokinesis (cytochalasin B, cytochalasin D or nocodazole) to examine the toxicity of these inhibitors on cloning. All of the oocytes exposed to nocodazole-containing media formed many small pseudo-pronuclei, whereas with cytochalasin-containing media most of the activated oocytes formed only two pseudo-pronuclei. Despite such differences, 42--61% of reconstructed embryos developed to the morula-blastocyst stage and 1--3% developed to full term in all groups. Addition of 1% (v/v) DMSO to the activation medium significantly improved the frequency of development to the blastocyst stage and full term; however, this improvement did not lead to a higher success rate in the generation of live cloned offspring. These results show that activated mouse oocytes/zygotes are not effective cytoplasmic recipients with the methods described and that the lack of success of cloning is not due to inhibition of cytokinesis.
A novel murine tryptase involved in blastocyst hatching and outgrowth
Authors: O'Sullivan CM , Rancourt SL , Liu SY , Rancourt DE
Published: March 2004

Before implantation the blastocyst is maintained within a proteinaceous coat, the zona pellucida, which prevents polyspermy and ectopic pregnancy. An extracellular trypsin-like activity, which is necessary for hatching from the zona pellucida in vitro, is localized to the abembryonic pole of the blastocyst. Upon hatching, the extracellular matrix-degrading proteinases urokinase plasminogen activator (uPA) and matrix metalloproteinase 9 (MMP-9) are thought to promote blastocyst invasion. However, gene disruption experiments have demonstrated that uPA and MMP-9 are dispensable and, thus, that other key enzymes are involved in implantation. In this study, a novel implantation serine proteinase (ISP1) gene, which is distantly related to haematopoietic tryptases and represents a novel branch of the S1 proteinase family, was cloned. ISP1 is expressed throughout morulae and blastocysts during hatching and outgrowth. Abrogation of ISP1 mRNA accumulation using antisense oligodeoxynucleotides disrupts blastocyst hatching and outgrowth in vitro. The results of this study indicate that the ISP1 gene probably encodes the long sought after 'hatching enzyme' that is localized to the abembryonic pole during hatching in vitro. ISP1 is the earliest embryo-specific proteinase to be expressed in implantation and may play a critical role in connecting embryo hatching to the establishment of implantation competence at the abembryonic pole of the blastocyst.
Immunolocalization of progesterone receptors in the canine ovary and their relation to sex steroid hormone concentrations
Authors: Vermeirsch H , Simoens P , Coryn M , Van den Broeck W
Published: March 2004

The aim of the present study was to describe the normal cellular distribution of progesterone receptors in the canine ovary at different stages of the oestrous cycle. Samples of both ovaries were obtained from 75 healthy adult bitches of various breeds and ages, including five pregnant bitches and three bitches that had just delivered. The presence of progesterone receptors was visualized by immunohistochemistry on paraffin wax sections using a monoclonal antibody. Nuclear staining for progesterone receptors was observed in the surface epithelium, cortical tubules, rete ovarii, follicle cells, thecal cells, luteal cells, granulosa cell cords and ovarian stroma. The staining intensity for progesterone receptors in the follicle cells increased with the stage of follicle development, indicating an intrafollicular role of progesterone in the mechanism of ovulation and luteinization. The stronger staining intensities for progesterone receptors in thecal cells compared with follicle cells may be explained by the fact that thecal cells mediate some effects of steroid hormones on the follicle cells in secondary and tertiary follicles. Little correlation was found between the expression of progesterone receptors in follicle cells and oestradiol, progesterone or testosterone concentrations. This finding indicates a different regulating mechanism for progesterone receptors in canine ovarian follicles compared with other tissues of the genital tract. During pregnancy all groups of ovarian cells had lower staining intensity scores than during the oestrous cycle, although the sex steroid hormone concentrations in pregnant bitches were similar to those in non-pregnant bitches during the luteal phase of the oestrous cycle. The lower expression of progesterone receptors during pregnancy may be due to higher tissue concentrations of progesterone that are not reflected in the serum because of haemodilution and increased metabolism and clearance during pregnancy.
Role of vascular endothelial growth factor in endometrial vascular events before implantation in rats
Authors: Rabbani ML , Rogers PA
Published: March 2004

Two endometrial vascular events, increased permeability and endothelial cell proliferation, characterize the early stages of embryo implantation in rats. Vascular endothelial growth factor (VEGF), a heparin-binding homodimeric glycoprotein, is expressed in the uterus at the time of implantation. The aim of this study was to investigate the effects of systemic administration of blocking antibody to VEGF on endometrial vascular permeability and endothelial cell proliferation around the time of embryo implantation in rats. Rats were injected i.p. with 1.0, 0.8 or 0.6 ml of anti-VEGF antibody or control serum on day 3 of pregnancy and Evans blue dye was administered i.v. on day 5 of pregnancy. The number of implantation sites (blue bands along the uterus) was counted and endothelial cell proliferation was identified using double staining immunohistochemistry. The number of blue bands was significantly lower (P < 0.05) after injection of 1.0 ml blocking antibody (1.8 +/- 1.56) compared with that in control rats (11.6 +/- 1.97). The number of blue bands was significantly different among rats injected with 1.0 (1.8 +/- 1.56), 0.8 (6.0 +/- 3.67) or 0.6 (10.7 +/- 0.33) ml anti-VEGF antibody, indicating a concentration effect of anti-VEGF antibody. No significant differences in endothelial cell proliferation at embryo implantation sites were observed after injection with anti-VEGF antibody or control serum. At intersites (uterine zones between the blue bands), injection of 0.6 ml anti-VEGF antibody caused a significant reduction (P < 0.05) in the percentage of vessels with proliferating endothelial cells (7.25 +/- 2.39%), with a non-significant reduction using 1.0 or 0.8 ml anti-VEGF antibody. No significant differences in microvascular density at either embryo implantation sites or intersites were observed between rats injected with anti-VEGF antibody or control serum. In conclusion, VEGF is the main factor responsible for increased endometrial vascular permeability at implantation. It may regulate endometrial endothelial cell proliferation before implantation and at intersites, thus indicating that factors or mechanisms other than VEGF may regulate endothelial cell proliferation at the site of implantation.
Effect of LH on circulating oestradiol and follicular fluid factor concentrations during follicle deviation in cattle
Authors: Ginther OJ , Bergfelt DR , Beg MA , Kot K
Published: March 2004

Progesterone was used to reduce LH concentrations starting at the time when the largest follicle was > or = 5.7 mm in diameter or well before the expected start of follicle deviation (largest follicle > or = 8.5 mm in diameter). Plasma concentrations of LH, FSH and oestradiol were determined at 4 h intervals in control and progesterone-treated heifers (n = 8 per group). Concentrations of LH were lower (P < 0.05) in the progesterone-treated group, reflecting an absence of the transient increase in LH concentrations that encompasses follicle deviation. An increase in oestradiol and a continued decrease in FSH occurred at the start of follicle deviation in the control cows but not in the treated heifers. In a second experiment, follicular fluid of the two largest follicles of control and progesterone-treated heifers was sampled at the expected start of deviation (n = 8--10 per group). The concentrations of oestradiol, but not androstenedione and free insulin-like growth factor I (IGF-I), in follicular fluid were higher (P < 0.001) in the largest follicle than in the second largest follicle. Progesterone treatment reduced (P < or = 0.02) the concentrations of all three factors in follicular fluid and increased (P < 0.05) the concentrations of insulin-like growth factor binding protein 2 (IGFBP-2). These results confirm that oestradiol contributes to the continued decrease in FSH concentrations after the start of follicle deviation. Prevention of the transient LH increase, the oestradiol increase and the continued FSH decrease did not significantly alter the mean time or follicle diameters characteristic of expected follicle deviation. However, in some treated individuals (three of eight), the observed follicle deviation was delayed. In addition, these results indicate that the secretion of oestradiol into the circulation and the increase in oestradiol and IGF-I and decrease in IGFBP-2 concentrations in the follicular fluid at the start of deviation are functions of the transient increase in LH concentrations that encompasses follicle deviation.
Differential expression of extracellular matrix components in the bovine oviduct during the oestrous cycle
Authors: Gabler C , Killian GJ , Einspanier R
Published: March 2004

Components of the extracellular matrix take part in tissue rebuilding as well as activating surface-bound growth factors. In the present study, expression and selected activities of urokinase-type plasminogen activator (uPA), matrix metalloproteinases (MMPs), their inhibitors (plasminogen activator inhibitor 1 (PAI-1) and tissue inhibitors of metalloproteinases (TIMPs)) were examined in bovine oviducts by RT--PCR, ribonuclease protection assay and activity assays. A high content of mRNA encoding for uPA was detected before ovulation with a three-fold decrease after ovulation. In contrast, PAI-1 expression appeared to be stable during the oestrous cycle. Oviductal flushings produced caseinolytic zones in zymograms containing plasminogen at approximately 50 kDa and 28 kDa. An activity assay for uPA showed highest net activity during the early to mid-luteal phase. Increased TIMP-1 and MMP-2 mRNA concentrations were found around the time of ovulation compared with the luteal phase. In contrast, MMP-1 mRNA transcripts were enriched during the early to mid-luteal phase. Gelatin zymograms detected a 70--72 kDa protease activity showing an oestrous cycle-dependent activity with highest activity before ovulation. Reverse zymography detecting TIMPs revealed proteins between 21 kDa and 24 kDa. Only for the smallest (21 kDa) protein were amounts increased around the time of ovulation compared with the luteal phase. The observation that several extracellular matrix components were regulated distinctly in bovine oviducts indicates that local interactions between these components, growth factors, gametes and the embryo are possible and may influence fertilization and early embryonic development.
Control of red fox (Vulpes vulpes) fertility with cabergoline: dose response and timing of intervention
Authors: Marks CA , Jalkanen L , Savolainen R , Short RV
Published: March 2004

Cabergoline, a potent dopamine agonist and inhibitor of prolactin secretion, was investigated as a potential fertility control agent in the red fox ( Vulpes vulpes). Sixty silver fox vixens were selected randomly and artificially inseminated. Cabergoline was fed to groups of 12 vixens in a minced beef ration either as a single dose of 25, 50 or 100 microg kg(-1), or a dose of 50 microg kg(-1) that was repeated 2 days later (2 x 50 microg kg(-1)). Four foxes from each group of 12 were given cabergoline at day 28, day 35 or day 48 after artificial insemination, and a control group of four foxes was used as a comparison for each dose day. In a separate trial, two groups of five foxes were selected randomly from the farm population and fed 100 microg kg(-1) of either cabergoline or a placebo each day from day 42 to day 46 of pregnancy. Foxes that received single doses of cabergoline of 100 microg kg(-1) or 2 x 50 microg kg(-1) aborted at day 28, but the same doses did not result in abortions when administered on days 35 and 48. Although lactation was not terminated in groups that received a single or double dose of cabergoline, increased post-natal cub mortality was associated with cabergoline administration. Growth of cubs between 4 and 8 weeks of age was not inhibited in vixens that received cabergoline. Doses of 100 microg cabergoline kg(-1) administered each day from day 42 to day 46 resulted in abortions and terminated lactation. The capacity of single doses of cabergoline to cause abortions in the red fox during mid- rather than late pregnancy is contrary to reported observations for the domestic dog. This finding indicates that luteotrophic support of the corpus luteum by prolactin may be more important at mid-pregnancy in the red fox. The results of this study support previous field observations that cabergoline delivered in bait affects the reproductive success of vixens and may be a practical adjunct to the lethal control of wild red foxes in Australia.
Glycoconjugates on the surface of epididymal spermatozoa in a marsupial, the brushtail possum, Trichosurus vulpecula
Authors: Cooper NJ , McClean RV , Leigh CM , Breed WG
Published: March 2004

Variation in localization and distribution of saccharides on the sperm surface of a marsupial, the brushtail possum, Trichosurus vulpecula, was compared between spermatozoa from the caput and cauda epididymides. Spermatozoa were subjected to the following treatments: (i) unfixed and fixed spermatozoa were stained with fluorescein-labelled lectins; (ii) unfixed spermatozoa were incubated with lectins for determination of agglutination; and (iii) spermatozoa were incubated with detergent to remove the plasmalemma, the glycoproteins were separated on SDS-PAGE and western blots were stained with biotinylated lectins. Many of the fluorescein isothiocyanate (FITC)-labelled lectins bound selectively to the sperm surface, and marked differences were found in lectin staining affinity between caput and cauda epididymal spermatozoa. Incubation of spermatozoa from the cauda epididymidis with neuraminidase reversed many of the differences in staining of the cauda epididymal spermatozoa, indicating masking of some terminal saccharides by sialic acid. Agglutination of spermatozoa from the caput epididymidis occurred after incubation with Concanavalin A (ConA) and soybean agglutinin (SBA), but agglutination was less extensive for spermatozoa from the cauda epididymidis. Western blot analysis indicated several ConA-positive bands in caput sperm extracts, but fewer positive bands in the cauda sperm extracts, whereas SBA stained four bands from caput but none from the cauda epididymal spermatozoa. These results demonstrate extensive glycosylation of the surface proteins of spermatozoa from the caput epididymidis and significant differences in spermatozoa from the cauda epididymidis. In general, the findings indicate similar glycosylation of the surface of marsupial spermatozoa to those from eutherian mammals despite marked differences in their morphology and early divergence of marsupials from eutherian mammals. It would appear that this situation differs markedly from that in sub-mammalian vertebrates.
Activation of cumulus-free equine oocytes: effect of maturation medium, calcium ionophore concentration and duration of cycloheximide exposure
Authors: Choi YH , Love CC , Varner DD , Thompson JA , Hinrichs K
Published: March 2004

Two different culture media (TCM-199 and follicular fluid), two activation treatments (10 and 50 micromol calcium ionophore l(-1)) and three culture periods with cycloheximide were evaluated to find effective culture conditions for activation of cumulus-free equine oocytes. Oocytes were collected by scraping the follicle walls of ovaries obtained from an abattoir. Oocytes with expanded cumuli were matured at 38.2 degrees C in a humidified atmosphere of 5% CO(2) in air, in either TCM-199 with 10% fetal bovine serum (FBS) and 5 microU FSH ml(-1), or in 100% follicular fluid derived from a preovulatory follicle 24 h after injection of hCG. After 40--42 h of in vitro maturation, oocytes were denuded by gentle pipetting in TCM-199 plus 10% FBS with hyaluronidase. Oocytes with intact cytoplasmic membranes (n = 398; 94% presumed metaphase II) were treated in protein-free PBS with 10 or 50 micromol calcium ionophore l(-1) for 5 min. After washing, the oocytes were cultured in TCM-199 containing 10% FBS and 10 microg cycloheximide ml(-1) for 6 h, in cycloheximide for 6 h and then in cycloheximide-free medium for 18 h, or in cycloheximide for 24 h. The oocytes were fixed and evaluated by fluorescence microscopy. Oocytes with pronucleus I--II (dense to decondensing chromatin), pronucleus III--IV (decondensed chromatin) or progressing towards the first cleavage division were considered activated. The activation rate for oocytes matured in TCM-199 was significantly (P < 0.05) higher than for oocytes matured in follicular fluid (49% (99/204) versus 35% (60/171), respectively; P < 0.05). Culture with cycloheximide for 24 h resulted in a significantly higher rate of activation (67%, 74/111) than did the 6 h (33%, 44/136) or 6 h plus 18 h (32%, 41/128) treatments. The highest rate of activation (82%) was observed in oocytes matured in TCM-199, treated with 50 micromol calcium ionophore l(-1) and cultured with cycloheximide for 24 h.
Genetic conflict, genomic imprinting and establishment of the epigenotype in relation to growth
Authors: Moore T
Published: March 2004

Genomic imprinting is the process that differentially modifies the parental alleles at certain genetic loci in the parental germlines. Such modifications of DNA and chromatin are somatically heritable and cause unequal expression of the parental alleles during subsequent development. In mammals, imprinted genes encode a relatively small number of functionally heterogeneous proteins. Nevertheless, imprinted genes exert important effects, primarily on fetal development, and their deregulation is implicated in a variety of pathologies including sporadic, inherited and induced growth disorders. Imprinted loci show several unusual structural and functional characteristics that may be related to mechanistic aspects of mono-allelic expression or to modes of evolution of imprinted genetic loci. Typically, imprinted genes are clustered in certain genomic regions and have relatively reduced intronic DNA content relative to non-imprinted genes. In addition, their regulatory regions frequently contain a combination of features including tandem repeats associated with differentially methylated CpG islands and overlapping transcription of coding or non-coding RNAs. The evolution of imprinting can be understood as the stable outcome of sexual selection acting differently on the parental alleles of genes that influence parental investment in offspring. Consistent with this explanation, imprinted genes are expressed predominantly during embryonic and postnatal development in mammals and in the developing endosperm of plants, and maternal or paternal expression at imprinted loci is associated with reduced or increased parental investment, respectively. Such selective forces have implications for understanding mechanistic aspects of genome reprogramming in the early mammalian embryo.
Intraovarian actions of oestrogen
Authors: Rosenfeld CS , Wagner JS , Roberts RM , Lubahn DB
Published: March 2004

Oestrogen regulates several hypothalamic and pituitary hormones, which in turn control ovarian functions. Oestrogen and its metabolites, such as catecholoestrogens, also have direct effects within the ovary. This review examines the roles of oestrogen in regulating ovarian folliculogenesis, ovulation and corpus luteum formation. Oestrogen promotes follicular development, which culminates in ovulation, by potentiating follicular development, granulosa cell expression of gonadotrophin receptors, steroidogenesis, and gap junction formation by granulosa cells, and by inhibiting granulosa cell apoptosis. In addition, oestrogen may be needed for corpus luteum formation and maintenance. Studies on mutant mice that either lack one or both of the known oestrogen receptors or are unable to synthesize oestrogen support some but not all of these prior inferences of the roles of oestrogen within the ovary. Although these transgenic mice have proved useful in determining some of the intraovarian actions of oestrogen, they present confounding problems, including hormonal imbalances, that hinder interpretation. Transgenic mice with conditional or tissue-directed mutations in their oestrogen receptors are needed to dissect the ovarian actions of oestrogen further. In addition, microarray technologies, combined with specific hormone treatment regimens are likely to provide an attractive, alternative approach to using mutant mice in clarifying the direct actions of oestrogen in the ovaries of other species.
Differential effects of prostaglandin F2alpha on in vitro luteinized bovine granulosa cells
Authors: Tsai SJ , Wiltbank MC
Published: March 2004

Prostaglandins have been implicated in various aspects of ovarian function including ovulation and luteolysis. In this study, the expression and regulation of inducible prostagland in G/H synthase (PGHS-2) and PGF(2alpha) receptors were investigated in bovine granulosa cells at various stages of differentiation. Firstly, the induction of PGF(2alpha) receptor mRNA and PGHS-2 mRNA in preovulatory granulosa cells was evaluated. Granulosa cells were collected from preovulatory follicles and cultured for 1, 4, 7 or 10 days. Cells were treated with hCG (10 iu) or with increasing doses of forskolin (0-10 micromol l(-1)) for 24 h. Forskolin increased steady-state concentrations of mRNA for PGHS-2 (> 20-fold) and PGF(2alpha) receptor (> 1000-fold) in a dose-dependent fashion. Use of selective protein kinase A inhibitor (H89) reduced both hCG- and forskolin-induced expression of PGF(2alpha) receptor mRNA and PGHS-2 mRNA. The hypothesis that luteinized granulosa cells would acquire PGF(2alpha) responsiveness similar to responses to PGF(2alpha) observed in vivo was also evaluated. Treatment with PGF(2alpha) (100 nmol l(-1)) reduced forskolin-induced expression of PGF(2alpha) receptor mRNA on days 4, 7 and 10, but not on day 1 of culture (n = 3). Treatment with PGF(2alpha) did not change forskolin-induced expression of PGHS-2 mRNA on or before day 4 of culture. In contrast, PGF(2alpha) significantly increased PGHS-2 mRNA expression in granulosa cells primed with forskolin for 7 or 10 days. In conclusion, expression of PGHS-2 and PGF(2alpha) receptor mRNA is protein kinase A-dependent in preovulatory bovine granulosa cells. Granulosa cells become PGF(2alpha)-responsive soon after expression of PGF(2alpha) receptor, whereas further differentiation is required before PGF(2alpha) induces PGHS-2 mRNA upregulation. These results demonstrate that at least two key transitions are required in PGF(2alpha)-induced luteal regression in the mid-cycle corpus luteum.
Reduction of long-term effects of local heating of the testis by treatment of rats with a GnRH agonist and an anti-androgen
Authors: Setchell BP , Ploen L , Ritzen EM
Published: March 2004

Heating the testes of anaesthetized adult rats to 43 degrees C for 30 min in a waterbath was followed by a large decrease in testis and epididymis mass and number of spermatozoa 35 days later. These parameters had recovered to some extent, but not completely, by days 70 and 97 after heating, but had decreased again in rats examined on day 182. There were no consistent effects of heating on androgen status, as determined by the concentrations of testosterone in blood and testis fluids, or by seminal vesicle mass, and interstitial fluid volume was increased in the heated testes. Treatment of rats with an implant of a GnRH agonist and daily injections of an anti-androgen for 14 days (sufficient in itself to cause large temporary decreases in tissue mass, number of spermatozoa and androgen status) did not reduce the initial decrease in testis mass or number of spermatozoa seen after heating, but reduced the later decreases in mass and number of spermatozoa significantly. These findings indicate that, as well as causing damage to spermatocytes and spermatids, as previously reported, heating also reduces the ability of spermatogonia to repopulate the seminiferous tubules at longer intervals after heating. Furthermore, it appears that this effect on the spermatogonia can be reduced by treating the animals with a GnRH agonist and anti-androgen, a treatment similar to that shown by other authors to improve recovery of the testis from irradiation or drug treatment.
Use of computer-assisted sperm motility assessment and multivariate pattern analysis to characterize ejaculate quality in Mohor gazelles (Gazella dama mhorr): effects of body weight, electroejaculation technique and short-term semen storage
Authors: Abaigar T , Cano M , Pickard AR , Holt WV
Published: March 2004

Subjective and objective semen assessments were performed on 18 male Mohor gazelles (Gazella dama mhorr). Sperm motility assessments combined with sperm plasma membrane and acrosomal integrity evaluations were undertaken as part of a captive breeding programme. The primary objective was to test methodology for short-term preservation of gazelle semen for artificial insemination (storage in N-[Tris(hydroxymethyl)methyl]-2-aminoethanesulphonic acid-Tris diluent (TEST) for up to 96 h at 17 degrees C). However, the secondary objective was to investigate phenotypic and genotypic influences on semen quality within this small population, which was established in 1971 with only 12 genetic founders. Sperm motility was measured by computer-assisted semen assessment and the data were analysed using a pattern analysis technique to detect and quantify naturally occurring sperm subpopulations within the semen samples. Four sperm subpopulations distinguishable by their motion characteristics were detected. The relative frequencies of two subpopulations (population 2: highly motile, non-linear; and population 4: poorly motile, non-linear) in fresh semen were correlated with the maximum voltage used during electroejaculation. The frequency of subpopulation 2 was negatively correlated with maximum voltage (r = -0.875, P < 0.0001) and the frequency of subpopulation 4 was positively correlated (r = 0.727, P < 0.005). The frequencies of all subpopulations varied significantly among the animals sampled (chi-squared = 2577.6, degrees of freedom = 54, P < 0.0001) and subpopulation 4 was also correlated with body weight (r = -0.59, P < 0.005). Semen stored at 17 degrees C retained motility, plasma membrane and acrosomal integrity for 48 h, but these measures decreased thereafter. The frequency of a sperm subpopulation showing uncoordinated but active motility increased significantly over the first 48 h and then decreased.
Immunohistochemical study of the distribution of adrenergic and peptidergic innervation in the equine uterus and the cervix
Authors: Bae SE , Corcoran BM , Watson ED
Published: March 2004

Little is known about neurogenic regulation of uterine contractility in mares. The present study investigated the distribution of adrenergic and peptidergic nerves in the mare uterus. Samples from the uterine horn, body and cervix were collected from 18 cyclic mares for immunohistochemistry. The uterus was well supplied with adrenergic nerves. A large number of tyrosine hydroxylase- and dopamine beta-hydroxylase-immunoreactive nerve bundles and fibres were present in the myometrium and endometrium in all regions of the uterus and cervix. These adrenergic nerve bundles and fibres travelled parallel to the muscle layers and were often associated with blood vessels. The density of peptidergic nerves was less than that of adrenergic nerves, but the pattern of distribution was similar. Neuropeptide Y-immunoreactive nerve fibres were the most abundant, whereas vasoactive intestinal polypeptide- and calcitonin gene-related peptide-immunoreactive nerve fibres were less frequently seen. Substance P-immunoreactive nerve fibres were the most sparse. Peptidergic nerves were distributed among the smooth muscle layers and near endometrial glands and were often associated with blood vessels in all regions of the uterus. The density of peptidergic nerve fibres was similar in the uterine horn and body but was slightly denser in the cervix. These findings indicate that uterine innervation may have an important role in controlling reproductive functions in mares.
Successful non-surgical deep intrauterine insemination with small numbers of spermatozoa in sows
Authors: Martinez EA , Vazquez JM , Roca J , Lucas X , Gil MA , Parrilla I , Vazquez JL , Day BN
Published: March 2004

A 100-fold reduction of the standard dose for artificial insemination in pigs (3 x 10(9) spermatozoa in 80-100 ml fluid) can be used when spermatozoa are deposited surgically next to the uterotubal junction. The present study was performed to develop a technique for non-surgical deep intrauterine insemination in pigs without sedation of the animal. In Expt 1, sows were weaned, treated to induce oestrus and used to evaluate the difficulties involved in the insertion of a flexible fibre optic endoscope through the cervix and along the uterine horn. Deep uterine catheterizations were performed on each sow at 30-40 h after hCG treatment in the crate in which the animal was housed. The endoscope was inserted through an artificial insemination spirette, moved through the cervical canal and propelled forward along one uterine horn until the entire endoscope was inserted. In 30 sows (90.9%) no or minor difficulties were observed during insertion and in these animals the procedure was completed in 4.1 +/- 0.26 min. Insertion of the endoscope through the cervical canal was not possible in only one sow (3.03%). In Expt 2, endoscopic deep intrauterine insemination at 36 h after hCG treatment was performed in 15, 18 and 13 sows with 100, 20 or 5 x 10(7) spermatozoa, respectively, resulting in farrowing rates of 86.6%, 88.9% and 92.3%, respectively; there were no significant differences among groups. Farrowing rates after deep intrauterine inseminations were also not different from those achieved after standard intracervical insemination with 3 x 10(9) spermatozoa (control group: n = 48; 87.5%). Mean litter size (9.41 +/- 0.38 to 10.02 +/- 0.25) was also similar among the different experimental and control groups. In conclusion, endoscopic non-surgical deep intrauterine inseminations can be performed quickly in sows, and normal farrowing rates and litter sizes can be obtained after insemination with a small number of spermatozoa.
Carbohydrate mediation of boar sperm binding to oviductal epithelial cells in vitro
Authors: Green CE , Bredl J , Holt WV , Watson PF , Fazeli A
Published: March 2004

After mating, mammalian spermatozoa are transported to the lower oviductal isthmus. Spermatozoa are sequestered at the isthmus by attaching and interacting with oviductal epithelial cells, hence forming a sperm reservoir. In several mammalian species, specific carbohydrates mediate sperm-oviductal epithelial cell binding. A quantitative in vitro free cell bioassay was developed to investigate the involvement of carbohydrate recognition in pig sperm-oviductal epithelial cell interactions. This assay was validated. The sensitivity of the assay was such that it was possible to discriminate between different sperm concentrations and sperm-oviductal epithelial cell co-incubation periods, spermatozoa with damaged plasma membranes and epithelial cells of non-reproductive origin. Optimal conditions were used to incubate spermatozoa and oviductal epithelial cells in the presence of six hexose sugars at concentrations of 0, 2, 10 and 50 mmol l(-1). A significant (P < or = 0.05) reduction in the binding of spermatozoa to the oviductal epithelium was detected with 2, 10 and 50 mmol maltose l(-1), 50 mmol lactose l(-1) and 50 mmol mannose l(-1). These findings support the hypothesis that attachment of pig spermatozoa to oviductal epithelium before fertilization is mediated by carbohydrate recognition.
Function and localization of oxytocin receptors in the reproductive tissue of rams
Authors: Whittington K , Assinder SJ , Parkinson T , Lapwood KR , Nicholson HD
Published: March 2004

Oxytocin is present in the male reproductive tract and has been shown to increase contractility in the epididymis and to modulate steroidogenesis. This study investigated the effects of oxytocin in the testis in vivo, and the presence and cellular localization of oxytocin receptors in the reproductive tract of rams. During the breeding season, mature rams underwent efferent duct ligation before injection of either oxytocin (20 microg) or oxytocin plus an oxytocin antagonist (20 microg) into the testicular artery; the contralateral testicular artery received saline. Injection of oxytocin caused a significant increase (P < 0.05) in the concentration of spermatozoa collected from the rete testis. This effect was not observed after treatment with the oxytocin antagonist plus oxytocin. Western blot analysis performed using a specific oxytocin receptor antibody (020) identified a single immunoreactive band of 66 kDa in testicular and epididymal tissue. This band was present in uterine tissue but not in liver or muscle. Immunocytochemistry identified oxytocin receptors on Leydig and Sertoli cells of the testis, on epithelial cells throughout the epididymis, on peritubular smooth muscle cells in the cauda epididymidis, and on the epithelial cells and circular smooth muscle layer of the ductus deferens. These findings indicate that oxytocin can modulate sperm transport in the ram testis. A role for oxytocin in promoting sperm transit is supported by the localization of oxytocin receptors in the cauda epididymis and ductus deferens, and the presence of receptors on Leydig, Sertoli and epididymal epithelial cells provides further evidence that oxytocin may be involved in the local regulation of steroidogenesis.
Role of quinine-sensitive ion channels in volume regulation in boar and bull spermatozoa
Authors: Petrunkina AM , Harrison RA , Hebel M , Weitze KF , Topfer-Petersen E
Published: March 2004

The ability to reverse swelling caused by hypo-osmotic stress is an important cell function; in spermatozoa, it is likely to be of consequence during ejaculation and also during the thawing process that terminates cryopreservation. In this study, the time course of boar and bull sperm volume changes after exposure to hypo-osmotic conditions at 39 degrees C was recorded. Cell volume measurements of washed sperm suspensions were performed electronically in Hepes-buffered saline solutions of 300 and 180 mosmol kg(-1) containing 2.5 mmol K(+) l(-1). Treatment with quinine in the presence or absence of the potassium ionophore valinomycin was used to determine whether potassium channels were involved in the reversal of swelling. After exposure to hypo-osmotic conditions, both bull and boar spermatozoa showed initial swelling (up to 200% and 140% of initial volume, respectively, within 5 min), which was subsequently partially reversed (to about 150% and 120%, respectively, after 20 min). Incubation with quinine led to an increase in swelling in both species. However, bull sperm volume was already maximal (up to 294%) after 30 s and declined thereafter, whereas boar sperm volume increased slowly to a maximum of about 220% after 20 min. Valinomycin treatment caused quinine-induced swelling in bull spermatozoa to decrease rapidly to control (no quinine, no valinomycin) values, whereas in quinine-treated boar spermatozoa it had an opposite, enhancing effect. Interpreting these results in the light of data from studies by others on a variety of cell types, it is proposed that swelling-activated potassium channels are involved in regulatory volume decrease in both species of spermatozoa, but that boar spermatozoa may contain fewer swelling-activated chloride channels than do bull spermatozoa.
Windows in early mammary development: critical or not?
Authors: Knight CH , Sorensen A
Published: March 2004

Two critical windows in mammary development have been proposed. The first arises from observations in rodents that nutrition during fetal and neonatal periods can affect mammary ductular outgrowth, subsequent proliferative activity and, eventually, tumorigenesis, that is, potentially it could have a long-term effect on pathological outcome (breast cancer) in women. The second similarly involves early diet, but in this case the outcome is phenotypic, in that dairy heifers reared too quickly during the peripubertal period subsequently show impaired udder development and reduced milk yield persisting throughout life. Most mammary development occurs during pregnancy, but this period is usually thought of only in terms of the immediate outcome for the subsequent lactation; it is not believed to be a critical window, at least in terms of lifetime mammary productivity. This review examines the evidence underlying these various claims and attempts to define the mechanisms involved, and also considers whether derangements occurring earlier in life (prenatally) could also have long-term consequences for physiological or pathological mammary development.
Influence of placentally mediated fetal growth restriction on the onset of puberty in male and female lambs
Authors: Da Silva P , Aitken RP , Rhind SM , Racey PA , Wallace JM
Published: March 2004

The onset of puberty in prenatally growth-restricted versus normally grown lambs of both sexes, born in April and housed under natural photoperiod, was examined. Singleton pregnancies were established and adolescent ewes were offered a high or moderate nutrient intake throughout gestation. Placental mass was reduced (P < 0.001) in high compared with moderate intake dams and resulted in the birth of growth-restricted and normal birth weight offspring, respectively. At birth, female lambs weighed 3.43 kg versus 5.03 kg (P < 0.001; n = 14 per group) and male lambs weighed 2.75 kg versus 5.18 kg (P < 0.001; n = 7 per group) in growth-restricted and normal birth weight groups, respectively. Lambs suckled for 12 weeks and thereafter were fed ad libitum until week 43 of age. Growth-restricted lambs had lower preweaning live weight gains and this difference was more pronounced in male (P < 0.05) than in female lambs (P = 0.07). Thereafter, live weight remained lower (P < 0.05) in growth-restricted than in normally grown lambs of both sexes until week 25 of age. In females, the time of onset of puberty was similar in the two groups. All females ovulated and there were no differences in the number of ovarian cycles recorded or in the incidence of aberrant ovarian function. In males, testosterone concentrations and testicular volume were lower in growth-restricted compared with normally developed lambs from birth until weeks 28 and 35 of age, respectively (P < 0.05). The seasonal increase in plasma testosterone concentrations occurred later in growth-restricted than in normally developed lambs (P < 0.01) but the timing of maximum peak concentrations was similar. Peak testosterone concentrations were lower (P < 0.05) in growth-restricted than in normal male lambs.
Gender differences in the inhibitory effects of a reduction in ambient temperature and a reduction in food quantity on reproduction in the Southern African rodent, Rhabdomys pumilio
Authors: Jackson C , Bernard RT
Published: March 2004

The effects of a reduction in ambient temperature (from 26 degrees C to 15 degrees C) and a 10% reduction in daily food consumption on reproductively active male and female four-striped field mice ( Rhabdomys pumilio) were investigated. In male R. pumilio, both reduced ambient temperature and a reduction in food quantity had an inhibitory effect on spermatogenesis and on size of the reproductive organs, and this was greatest when the two factors were combined and the effect of fat was removed. Female R. pumilio responded differently and reproduction was inhibited by a reduction in food quantity, irrespective of ambient temperature. The masses of the ovaries and uterus, the numbers of developing follicles and corpora lutea, and the development of the uterine wall were all reduced by food deprivation at 26 degrees C to levels similar to those that resulted from a reduction in ambient temperature to 15 degrees C with a reduction in food quantity. It is concluded that reproduction in R. pumilio from the Eastern Cape Province of South Africa is opportunistic, that reproduction will be inhibited by an energetic challenge and that there is sexual dimorphism in the response to ambient temperature and food supply.