Showing papers in "Reproduction in 2001"

Oocyte control of ovarian follicular development and function in mammals.

Abstract: 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.

Oxidative stress, DNA damage and the Y chromosome.

Abstract: Recent advances in understanding of male infertility have implicated two major causative factors, oxidative stress and Y chromosome deletions. A major cause of oxidative stress appears to be the high rate of reactive oxygen species generation associated with the retention of excess residual cytoplasm in the sperm midpiece. Other possible causes include the redox cycling of xenobiotics, and antioxidant depletion or apoptosis. Oxidative stress induces peroxidative damage in the sperm plasma membrane and DNA damage in both the mitochondrial and nuclear genomes. Nuclear DNA damage in the germ line of the father may be associated with pathology in the offspring, including childhood cancer and infertility. Gene deletions on the non-recombining region of the Y chromosome account for the infertility observed in about 15% of patients with azoospermia and 5-10% of subjects with severe oligozoospermia. The Y chromosome is particularly susceptible to gene deletions because of the inability of the haploid genome to deploy recombination repair in retrieving lost genetic information. Aberrant recombination, defective chromatin packaging, abortive apoptosis and oxidative stress may all be involved in the aetiology of DNA damage in the germ line. The factors responsible for Y chromosome deletions in spermatozoa remain unresolved but may be one facet of a central reproductive problem: controlling the amount of oxidative stress experienced by germ cells during their differentiation and maturation in the male reproductive tract.

Cellular basis for paracrine regulation of ovarian follicle development

Abstract: Paracrine factors secreted by oocytes and somatic cells regulate many important aspects of early ovarian follicle development in mammals. From activation of dormant primordial follicles to selection of secondary follicles, locally acting factors have been identified that appear to exert important effects on the growth and differentiation of oocytes and granulosa cells. This article summarizes evidence to support a model for bi-directional paracrine communication that is based on developmental regulation of the delivery and reception of paracrine factors at the oocyte-granulosa cell interface. Transzonal projections that originate from granulosa cells and terminate at the oocyte plasma membrane provide a polarized means to orient the secretory organelles of somatic cells. Characterization of transzonal projections in follicles from normal and genetically modified mice reveals dynamic changes in the density and stability of transzonal projections. On the basis of new data analysing the orientation and cytoskeletal content of transzonal projections in mammalian oocytes, a model is proposed for regulation of paracrine growth factor secretion by follicle-stimulating hormone. These findings have immediate implications for ovarian hyperstimulation protocols and follicle culture models as related to the production of mammalian embryos by assisted reproductive technologies.

Relationship between maternal endocrine environment, early embryo development and inhibition of the luteolytic mechanism in cows

Abstract: In this study, the relationship between maternal hormone environment and early embryo development in mature non-lactating Holstein-Friesian cows was investigated. Animals were inseminated at either 72 or 96 h after prostaglandin injection (n = 23) or were left as uninseminated controls (n = 10). Plasma samples were collected once a day from the first day of insemination (day 1) until day 16, when the cows underwent an oxytocin challenge, and were then slaughtered and their reproductive tracts removed. The tracts were flushed to collect embryos and the flushes were measured for interferon tau (IFN-tau) activity. Inseminated cows without an embryo on day 16 (n = 5) underwent both delayed ovulation (indicated by delayed decrease in oestradiol concentrations) and a delayed increase in progesterone concentrations after ovulation compared with cows with an embryo on day 16 (n = 15). Within the group of cows with an embryo, those with poorly developed embryos producing undetectable concentrations of IFN-tau (n = 7) had similar oestradiol profiles but underwent a delayed progesterone increase after ovulation compared with cows with well developed embryos producing measurable quantities of IFN-tau (n = 8). In the cows with an embryo, the plasma concentration of 13,14-dihydro-15-keto PGF2a, the principal metabolite of PGF2a, after injection of oxytocin was lower than that of control cows and cows without an embryo. However, when the cows with an embryo were compared on the basis of production of embryonic IFN-tau, the PGF2a response to oxytocin was attenuated completely in cows that had measurable IFN-tau activity, whereas a response of similar magnitude to that in control cows and cows without an embryo was observed in those with undetectable IFN-tau activities. In conclusion, the successful maternal recognition of pregnancy in cows depends on the presence of a sufficiently well developed embryo producing sufficient quantities of IFN-tau, which is, in turn, dependent on an appropriate pattern of maternal progesterone secretion.

Proliferation and differentiation of spermatogonial stem cells

Abstract: Spermatogonial stem cells (A s spermatogonia) are single cells that either renew themselves or produce A pr (paired) spermatogonia predestined to differentiate. In turn, the A pr divide into chains of A al (aligned) spermatogonia that also divide. The ratio between self-renewal and differentiation of the stem cells is regulated by glial cell line-derived neurotrophic factor produced by Sertoli cells, while the receptors are expressed in stem cells. A s , A pr and A al spermatogonia proliferate during part of the epithelial cycle forming many A al spermatogonia. During epithelial stage VIII, almost all A al spermatogonia, few A pr and very few A s spermatogonia differentiate into A1 spermatogonia. A number of molecules are involved in this differentiation step including the stem cell factor-c-kit system, the Dazl RNA binding protein, cyclin D 2 and retinoic acid. There is no fine regulation of the density of spermatogonial stem cells and consequently, in some areas, many A1 and, in other areas, few A1 spermatogonia are formed. An equal density of spermatocytes is then obtained by the apoptosis of A2, A3 or A4 spermatogonia to remove the surplus cells. The Bcl-2 family members Bax and Bcl-x L are involved in this density regulation. Several mechanisms are available to cope with major or minor shortages in germ cell production. After severe cell loss, stem cell renewal is preferred above differentiation and the period of proliferation of A s , A pr and A al spermatogonia is extended. Minor shortages are dealt with, at least in part, by less apoptosis among A2-A4 spermatogonia.

Maturation of human oocytes in vitro and their developmental competence

Abstract: Complete maturation of oocytes is essential for the developmental competence of embryos. Any interventions in the growth phase of the oocyte and the follicle in the ovary will affect oocyte maturation, fertilization and subsequent embryo development. Oocyte size is associated with maturation and embryo development in most species examined and this may indicate that a certain size is necessary to initiate the molecular cascade of normal nuclear and cytoplasmic maturation. The minimum size of follicle required for developmental competence in humans is 5-7 mm in diameter. Maturation in vitro can be accomplished in humans, but is associated with a loss of developmental competence unless the oocyte is near completion of its preovulatory growth phase. This loss of developmental competence is associated with the absence of specific proteins in oocytes cultured to metaphase II in vitro. The composition of culture medium used successfully for maturation of human oocytes is surprisingly similar to that originally developed for maturation of oocytes in follicle culture in vitro. The presence of follicle support cells in culture is necessary for the gonadotrophin-mediated response required to mature oocytes in vitro. Gonadotrophin concentration and the sequence of FSH and FSH-LH exposure may be important for human oocytes, particularly those not exposed to the gonadotrophin surge in vivo. More research is needed to describe the molecular and cellular events, the presence of checkpoints and the role of gene expression, translation and protein uptake on completing oocyte maturation in vitro and in vivo. In the meantime, there are very clear applications for maturing oocytes in human reproductive medicine and the success rates achieved in some of these special applications are clinically valuable.

Hyperactivation of mammalian spermatozoa: function and regulation

Abstract: Hyperactivation is a movement pattern observed in spermatozoa at the site and time of fertilization in mammals It may be critical to the success of fertilization, because it enhances the ability of spermatozoa to detach from the wall of the oviduct, to move around in the labyrinthine lumen of the oviduct, to penetrate mucous substances and, finally, to penetrate the zona pellucida of the oocyte The movement of hyperactivated spermatozoa appears different under different physical conditions and in different species, but basically it involves an increase in flagellar bend amplitude and beat asymmetry Presumably, there is a signal or signals in the oviduct to initiate hyperactivation at the appropriate time; however, none has yet been identified There is evidence that the source of the signal is follicular fluid, yet spermatozoa are known to hyperactivate before ovulation would release the fluid into the oviduct Although the signal transduction cascade regulating hyperactivation remains to be described completely, it is clear that calcium ions interact with the axoneme of the flagellum to switch on hyperactivation The process may also involve increases in intracellular cAMP, which at least is required to support motility in general Although hyperactivation usually occurs during capacitation, the two events are regulated by different pathways

Potential local regulatory functions of inhibins, activins and follistatin in the ovary.

Abstract: The changing pattern of granulosa cell expression of inhibin/activin subunits and follistatin during follicle development and their differential regulation by extrinsic and intraovarian factors supports evidence from functional studies, mostly in vitro, that these proteins have important roles in folliculogenesis, oocyte maturation and corpus luteum function. Gonadal inhibins function as negative feedback hormones to regulate the synthesis and secretion of pituitary FSH, a key determinant of follicle development, but there is little supportive evidence for a peripheral endocrine role for ovary-derived activins or follistatin in this regard. However, activins and follistatin are expressed in numerous other tissues, including anterior pituitary, and they are firmly implicated as local intrapituitary regulators of FSH secretion. Intraovarian actions of granulosa cell-derived activins include the promotion of granulosa cell proliferation and upregulation of FSH receptors, P450arom, oestrogen synthesis, granulosa cell LH receptors and enhancement of oocyte maturation. Through its activin-binding role, follistatin can reverse each of these activin-induced responses. In addition to their endocrine feedback role, granulosa-derived inhibins can sensitize theca cells to LH, thereby enhancing the production of androgens, an essential requirement for follicular oestrogen synthesis. Activins can oppose this effect and suppress thecal androgen production. Granulosa cells overproduce inhibin a subunit precursor relative to betaA/betaB subunit precursors and evidence indicates that different parts of the inhibin a subunit precursor have intrinsic biological activities distinct from inhibin alphabetaA/B dimer, and serve as additional local modulators of follicle and corpus luteum function.

Translocation of active mitochondria during pig oocyte maturation, fertilization and early embryo development in vitro

Abstract: The distribution of active mitochondria during pig oocyte maturation, fertilization and early embryo development in vitro was revealed by using MitoTracker Green staining and confocal laser scanning microscopy. The regulation of mitochondrial translocation by microfilaments and microtubules was also studied. In oocytes collected from small follicles, strong staining of active mitochondria was observed in the cell cortex. Accumulation of active mitochondria in the peripheral cytoplasm and around the germinal vesicles was characteristic of fully grown oocytes collected from large follicles. Mitochondria accumulated in the perinuclear area during meiotic progression from germinal vesicle breakdown (GVBD) to anaphase I. Larger mitochondrial foci were formed and moved to the inner cytoplasm in mature oocytes. Compared with the oocytes matured in vivo, in which large mitochondrial foci were distributed throughout the cytoplasm, mitochondria were not observed in the central cytoplasm in most of the oocytes matured in vitro. Strong staining of mitochondria was observed in the first polar bodies in metaphase II oocytes. In fertilized eggs, active mitochondria aggregated in the pronuclear region. Perinuclear clustering and a cortical ring were the most marked features of early cleavage. Active mitochondria were distributed in both inner cell mass cells and trophectoderm cells of the blastocysts. Disassembly of microtubules with nocodazole inhibited both mitochondrial aggregations to the germinal vesicle area and their inward movement to the inner cytoplasm during oocyte maturation, as well as the translocation of mitochondria to the peri-pronuclear region during fertilization, whereas disruption of microfilaments by cytochalasin B had no effects. These data indicate that: (i) oocyte maturation, fertilization and early embryo development in pigs are associated with changes in active mitochondrial distribution; (ii) mitochondrial translocation is mediated by microtubules, but not by microfilaments; and (iii) in vitro maturation conditions may cause incomplete movement of mitochondria to the inner cytoplasm and thus affect cytoplasmic maturation.

Seasonal changes in bovine fertility: relation to developmental competence of oocytes, membrane properties and fatty acid composition of follicles.

Abstract: Follicle dynamics and oocyte viability in Holstein primiparous and multiparous cows and the relationships between fertility and the biochemical and physical properties of oocyte membranes with season were examined. The conception rates of primiparous (n = 70 885) and multiparous (n = 143 490) cows differed, peaking in the winter and decreasing in the summer. The number of follicles 3-8 mm in diameter per ovary was higher in winter (19.6) compared with summer (12.0). However, in winter the percentage of ovaries with fewer than ten follicles per ovary was 16%, in contrast to 50% in summer. After aspiration of follicles, 7.5 oocytes per ovary were found in winter and 5.0 oocytes per ovary in summer. Cleavage to the two- to four-cell stage after chemical activation was greater in winter than in summer; this was enhanced at the morula stage and embryo development to the blastocyst stage was significantly higher in winter than in summer. Determination of the lipid phase transition in oocyte membranes revealed a shift of 6 degrees C between summer and winter. Fatty acid composition of phospholipids from follicular fluid, granulosa cells and oocytes indicated that there was a higher percentage of saturated fatty acids during the summer and that the percentages of mono-unsaturated and polyunsaturated fatty acids were higher in oocytes and granulosa cells during the winter. Oocytes and granulosa cells had similar fatty acid compositions, in contrast to follicular fluid. These results may explain the differences in the ability of oocytes to develop to the blastocyst stage at different seasons. Thus, temperature changes may lead to changes in membrane properties, which, in turn, can influence oocyte function and fertility.

Lipid and fatty acid analysis of fresh and frozen-thawed immature and in vitro matured bovine oocytes

Abstract: The lipid content and fatty acid composition of fresh immature and in vitro matured bovine oocytes cultured in media with or without serum, and also those of frozen-thawed immature oocytes were analysed. All oocytes were ranked (A or B) on the basis of their cytoplasmic quality. Fatty acid composition (mol %; w/w) in the total lipid fraction was analysed by gas chromatography. Triglyceride, total cholesterol, phospholipid (phosphocholine-containing phospholipid) and non-esterified fatty acid contents of immature and in vitro matured oocytes were determined using lipid analysis kits. Phosphocholine-containing phospholipid and non-esterified fatty acid contents were determined in frozen-thawed immature bovine oocytes. Palmitic acid was the most abundant fatty acid in immature oocytes (A: 35%, B: 36%), and in in vitro matured oocytes cultured in the medium containing serum (A: 36%, B: 35%) or polyvinyl alcohol (A: 33%, B: 36%). Oleic acid was the second most abundant fatty acid in all A ranked oocytes, whereas stearic acid was the second most abundant fatty acid in all B ranked oocytes. There were significant differences (P < 0.05) in linoleic and arachidonic acid fractions between A and B ranked immature oocytes. In vitro matured oocytes had significantly (P < 0.05) lower proportions of linoleic and arachidonic acids, and significantly (P < 0.01) lower contents of triglyceride and total cholesterol compared with those of immature oocytes. The fatty acid composition of in vitro matured oocytes cultured in medium containing fetal calf serum or polyvinyl alcohol was similar, but significant differences (P < 0.01) in triglyceride and the total cholesterol content were observed. There was a significant decrease (P < 0.05) in the arachidonic acid proportion in frozen-thawed immature oocytes compared with that in fresh immature oocytes. In addition, significant (P < 0.05) decreases in both phospholipid (15.8--10.6 pmol) and non-esterified fatty acid (11.0--4.1 pmol) were found in frozen--thawed immature oocytes. The results indicate that lipids are available for use as an energy source for maturation and that serum lipids are incorporated into the oocyte cytoplasm during in vitro maturation. The changes in the lipid content (mainly phospholipid) and fatty acid composition were also observed in frozen--thawed immature oocytes. The study indicates that the alteration of fatty acid composition in bovine oocytes might improve maturation and cryopreservation.

Fetal growth restriction: adaptations and consequences

Abstract: A range of pathophysiological factors can result in a perturbation or restriction of fetal growth, and the cardiovascular, neuroendocrine and metabolic adaptations of the fetus to these stimuli will depend on their nature, timing and intensity. The critical importance of these physiological adaptations for both immediate survival and long-term health outcomes has provided an impetus for experimental studies of the nature and consequences of specific fetal adaptations to a poor intrauterine environment. This review summarizes data from recent studies that have focused on the responses of the fetal cardiovascular, sympathoadrenal, hypothalamo-pituitary-adrenal and renin-angiotensin systems to experimental restriction of placental function in the sheep and discusses the consequences of these adaptations for fetal, neonatal and adult health.

Changes in sperm quality and lipid composition during cryopreservation of boar semen

Abstract: The effect of cryopreservation on boar sperm viability, motility, lipid content and antioxidant enzymatic activities was studied. Three classes of semen were determined according to a cluster analysis on the basis of the proportion of live and dead cells after freezing and thawing. The classes identified were: high (H, n = 4), average (A, n = 12) and low (L, n = 3) viability. The concentration of sperm cells decreased from class H to A to L. Fresh semen samples with higher viability and a higher proportion of motile cells also maintained better quality after the freezing and thawing procedure. Sperm viability and motility in both fresh and thawed samples were similar in classes H and A, while significantly lower values were measured in class L. The relative decrease in sperm viability and motility after cryopreservation increased from class H to A to L. The lipid content of spermatozoa (micrograms per 10(9) cells) increased significantly after freezing and thawing in classes H and A but not in class L. This result indicated that active sperm lipid metabolism might be responsible for the increase in lipid content. Phospholipid and triacylglycerol contents increased whereas free cholesterol content decreased after thawing. The fatty acid composition of fresh spermatozoa was similar in all three classes. The proportion of polyunsaturated fatty acids decreased significantly after freezing and thawing, indicating contamination from the diluent or peroxidation. After freezing and thawing, superoxide dismutase activity in spermatozoa was significantly higher in class L than in classes H and A, which did not differ from each other.

Formation of Fallopian tubal fluid: role of a neglected epithelium

Abstract: Fluid produced and secreted by the Fallopian tube provides the environment in which gamete transport and maturation, fertilization and early embryo development occur. This review describes the composition of oviductal fluid in terms of ions and nutrients such as glucose, lactate, pyruvate and amino acids. The function of oestrogen-specific glycoprotein is discussed. The mechanisms of fluid secretion and agents known to influence fluid production and secretion are described. Clinical implications of abnormal oviductal fluid production and secretion in hydrosalpinx and pelvic inflammatory disease are also discussed.

Cytokine control in human endometrium.

Abstract: Cytokines within endometrium participate in both menstruation and implantation but also contribute to the defence mechanisms of the mucosal epithelium. Endometrium is under the control of steroid hormones, particularly progesterone and, thus, control of cytokines by this steroid is important. Although appreciable numbers of progesterone receptors are not found in endometrial leucocytes, progesterone can modulate cytokines by acting on uterine cells expressing the receptor. The NFkappaB pathway is important in the control of cytokine synthesis and can modulate production of chemokines, matrix metalloproteinases and the inducible prostaglandin synthesis enzyme COX-2. NFkappaB activity can be inhibited by progesterone by either stimulating synthesis of IkappaB, the molecule that restrains NFkappaB in the cytosol, or after binding to the nuclear receptor, competing with NFkappaB for recognition sites on the relevant gene. In this way, progesterone can limit pro-inflammatory pathways. The major palliatives for endometrial dysfunctions such as menorrhagia and dysmenorrhoea have been the non-steroidal anti-inflammatory drugs that inhibit prostaglandin synthesis. Prostaglandins have major effects on cytokine production but the direct action of prostaglandin E on leucocytes is not a pro-inflammatory response but is to stimulate interleukin 10 and inhibit interleukin 12 synthesis. The likely effect of the non-steroidal anti-inflammatory drugs is on the cells surrounding the small blood vessels, where a synergistic action between prostaglandin and chemokine will induce leucocyte entry and activation leading to lysis of connective tissue and menstruation. At the time of implantation, tight control of cytokine synthesis is required. Although leukaemia inhibitory factor is essential to implantation, the mouse knockout models show that the prostaglandin system is also essential but that there are mutually supportive pathways that compensate for the knockout of many cytokines.

Aneuploidy in human spermatozoa : FISH analysis in men with constitutional chromosomal abnormalities, and in infertile men

Abstract: Reproductive difficulties are associated intimately with cytogenetic abnormalities. This article reviews multicolour fluorescence in situ hybridization studies on spermatozoa from men with constitutional chromosomal abnormalities and the consequences for spermatozoa, and on chromosomal abnormalities in the spermatozoa of infertile men who have normal somatic karyotypes. In 47,XYY men, the frequencies of 24,XY and 24,YY spermatozoa appear to be < or = 1%. Klinefelter (47,XXY) and mosaic Klinefelter patients had sperm aneuploidy frequencies of 2-25% and 1.5-7.0%, respectively. Robertsonian translocation carriers had 3-27% spermatozoa unbalanced for the chromosomes involved in the translocation, with a possible modest interchromosomal effect, but none of the increased frequencies of chromosomal disomy approached 1%. The frequency of chromosomally unbalanced spermatozoa in reciprocal translocations averages 50%, is strongly dependent on the chromosomes involved in the individual translocation, and may be slightly increased as a result of a small interchromosomal effect. Infertile men with a normal karyotype and low sperm concentration or certain types of morphologically abnormal spermatozoa have a significantly increased risk of producing aneuploid spermatozoa, particularly for the sex chromosomes. An increased risk of sperm aneuploidy was not observed in infertile men with poor sperm motility or in those with a normal karyotype and normal semen parameters.

Improvement of quality of oocytes collected in the autumn by enhanced removal of impaired follicles from previously heat-stressed cows.

Abstract: The fertility of dairy cows decreases during the summer and remains low during the cooler autumn although the animals are no longer under heat stress. The aim of this study was to characterize a delayed effect of summer heat stress on oocyte quality in the autumn and to improve oocyte quality by enhanced removal of follicles damaged during the previous summer. Lactating cows (n = 16) were subjected to heat stress during the summer. In autumn, ovarian follicles (3-7 mm in diameter) were aspirated by an ultrasound-guided procedure during four consecutive oestrous cycles. Follicles were aspirated from control cows on day 4 and from treated cows on days 4, 7, 11 and 15 of each oestrous cycle. All cows received PGF(2alpha) and GnRH injections on days 19 and 21, respectively, and maintained cyclicity, as indicated by plasma progesterone concentrations. On day 4 of each cycle, the oocytes recovered were examined morphologically, matured and activated in vitro, and cultured for 8 days. In cycle 1 (early October) both groups showed low percentages of grade 1 oocytes, cleavage, four- and eight-cell embryos, morulae and parthenogenetic blastocysts. Subsequently, the number of grade 1 oocytes increased earlier (cycle 2) in treated than in control cows (cycle 3; P < 0.05). The cleavage rate in the control group remained relatively low throughout (32-58%), whereas in the treated group it increased from 40% (cycle 1) to 75% (cycles 3 and 4; P < 0.05). The number at each stage of embryo development increased slightly but remained low throughout in the control group, whereas in the treated group significant (P < 0.05) increases of all stages were observed in cycles 3 and 4. The results show a delayed effect of summer heat stress on oocyte quality and embryo development in the autumn. Enhanced removal of the impaired cohort of follicles led to earlier emergence of healthy follicles and high quality oocytes in the autumn.

Development of cultured bovine embryos after exposure to high temperatures in the physiological range

Abstract: Embryonic development is inhibited by exposure of cultured embryos to high temperatures. However, culture temperatures used to demonstrate the effects of heat on development have been higher than the body temperatures experienced typically by heat-stressed cows. The aim of this study was to determine whether exposing bovine oocytes and embryos to temperatures characteristic of body temperatures of heat-stressed cows would affect embryonic development in vitro. The CO2 percentage of the gas phase was adjusted in all experiments to prevent pH changes in the medium caused by decreased solubility of CO2 at high temperatures. Fertilization of oocytes at 41.08C reduced cleavage rate and the percentage of oocytes that became blastocysts compared with at 38.58C. There was no deleterious effect of fertilization at 40.08C. When putative zygotes and two-cell embryos were exposed to a range of temperatures from 38.5 to 41.08C for 3, 6, 9 or 12 h, heat shock reduced the number that developed to the blastocyst stage but only after exposure to 41.08C for 9 or 12 h. In addition, it was tested whether low O2 tension would reduce the detrimental effects of heat shock. The deleterious effect of 41.08C was not dependent upon oxygen content or the gas mixture used for culture (5% versus 20.95% O 2 ), indicating that the deleterious effects of heat shock did not depend upon a high O 2 environment. In the final experiment, embryos were exposed to 24 h fluctuations in temperature designed to mimic the rectal temperatures of cows exposed to heat stress. Exposure of embryos to this pattern of temperatures starting after fertilization reduced development when embryos were exposed to this environment for 8 days but not when embryos were exposed for 1 day only. These findings indicate that embryonic development can be disrupted by a short-term severe or a prolonged mild heat shock and that the effects of heat shock are not artefacts of changes in pH or high oxygen tension.

Human endometrial angiogenesis.

Abstract: Angiogenesis is the development of new microvessels from existing vessels, a process that involves microvascular endothelial cells. Physiological angiogenesis rarely occurs in adults except in the ovary and endometrium during the reproductive life of females. Angiogenesis occurs by sprouting and non-sprouting mechanisms. Since endothelial sprouts are not observed in human endometrium, we hypothesized that non-sprouting mechanisms such as intussusception and elongation are involved in endometrial angiogenesis. The demand for angiogenesis differs spatially and temporally in the endometrium: angiogenesis occurs in the basalis layer during menstruation and in the functionalis and subepithelial capillary plexus during the proliferative and early secretory stages. Most studies have failed to demonstrate a link between expression of endometrial angiogenic factors and new vessel growth. However, we demonstrated recently a strong relationship between vascular endothelial growth factor (VEGF) immunolocalized in in-travascular neutrophils and endothelial cell proliferation in each of the subepithelial capillary plexus, functionalis and basalis regions of the human endometrium. Our data also indicate that focal neutrophil VEGF has a role in the development of the subepithelial capillary plexus and functionalis microvessels during the proliferative phase of the menstrual cycle. We propose that neutrophils are an intravascular source of VEGF for vessels that undergo angiogenesis by intussusception and elongation.

Alterations in development of reproductive and endocrine systems of wildlife populations exposed to endocrine-disrupting contaminants.

Abstract: Wildlife and human populations are affected by contaminants in natural settings. This problem has been a growing concern over the last decade with the realization that various environmental chemicals can alter the development and functioning of endocrine organs, cells and target tissues. Documented disruptions or alterations in reproductive activity, morphology or physiology in wildlife populations have been correlated with contaminant-induced modifications in endocrine system functioning. Alterations of the endocrine system are complex, and not limited to a particular organ or molecular mechanism. For instance, contaminants have been shown to (1) act as hormone receptor agonists or antagonists, (2) alter hormone production at its endocrine source, (3) alter the release of stimulatory or inhibitory hormones from the pituitary or hypothalamus, (4) alter hepatic enzymatic biotransformation of hormones, and (5) alter the concentration or functioning of serum-binding proteins, altering free hormone concentrations in the serum. This review focuses on two of these alterations, altered hormone synthesis and hepatic biotransformation, as a number of recent studies indicate that these actions are important components of endocrine disruption in developing organisms. The possible role of contaminants in altering sex determination mechanisms is also examined.

Comparison of the capacitation-like state of cooled boar spermatozoa with true capacitation

Abstract: Cryopreserved spermatozoa demonstrate reduced conception rates compared with fresh spermatozoa when used for artificial insemination. The preliminary stage of cryopreservation of spermatozoa involves cooling to 5 degrees C, during which spermatozoa experience a capacitation-like change, which may be partially responsible for the reduced conception rate observed. The aim of this study was to determine the nature of these capacitation-like changes and how much this process resembles true capacitation. Boar spermatozoa, cooled to 5 degrees C and re-warmed to physiological temperatures (39 degrees C), were compared with spermatozoa capacitated in Tyrode's complete medium (TALP) for 2 h at 39 degrees C. Fluorescent probes, and SDS-PAGE and western blotting were used to visualize events known to occur during capacitation in vitro. Chlortetracycline staining of membrane domains and Fluo-3 detection of changes in intracellular free calcium by flow cytometry in cooled and re-warmed spermatozoa showed similarities to those of capacitated spermatozoa. Alterations to lipid bilayer fluidity assessed by merocyanine fluorescence staining and intracellular signalling pathways detected by tyrosine phosphorylation of cooled and re-warmed spermatozoa, did not completely reflect the changes detected during capacitation in vitro. Thus, cooling spermatozoa to 5 degrees C results in a similar endpoint to that observed in capacitated cells in terms of reactive membranes and changes in intracellular ion concentrations, which may account for their comparable functionality. However, these modifications are not completely analogous and should not be considered true capacitation, but rather a by-passing of the capacitation process.

Delayed effect of heat stress on steroid production in medium-sized and preovulatory bovine follicles

Abstract: The low summer fertility of about 60% of the world dairycattle population is associated with high ambient tempera-tures. However, during the autumn, when air temperatureshave decreased and cows are no longer exposed to thermalstress, conception rates remain lower than in the winter(Hansen, 1997). This observation may be explained by thefacts that ovarian follicles are susceptible to heat stress(Badinga et al., 1993; Wolfenson et al., 1995) and that ittakes about 40–50 days for small antral follicles to developinto large dominant follicles (Lussier et al., 1987). Thus,exposure to summer heat stress during the early stages offollicular development may impair later follicular functionand decrease fertility in the autumn. Clear delayed effects ofsummer heat stress on follicular function have beenobserved: (i) a markedly low quality of oocytes collectedafter summer heat stress was associated with low develop-mental capability of embryos in vitroduring the autumn(Roth et al., 1999); and (ii) alterations in the pattern ofgrowth and development of medium-sized follicles asso-ciated with a marked increase in plasma FSH concentrationwere found during the first follicular wave of the oestrouscycle, subsequent to heat exposure (Roth et al., 2000a). Aseasonal study has shown that oestradiol concentration inthe follicular fluid and androstenedione production bythecal cells were both lower in dominant follicles collectedin autumn than in those collected in winter (Wolfenson etal., 1997). However, seasonal studies provided only limitedinformation regarding a delayed effect of heat stress onfollicular steroidogenesis owing to their multifactorialnature and the fact that proper contemporary controlanimals could not be used in such studies.The aim of the present study was to examine a possibledelayed effect of acute heat stress on follicular characteristics.Steroid production by granulosa and thecal cells was

Expression of oxytocin, oestrogen and progesterone receptors in uterine biopsy samples throughout the oestrous cycle and early pregnancy in cows

Abstract: This study examined the expression patterns of oxytocin and steroid receptors in the bovine endometrium during the oestrous cycle and early pregnancy to elucidate their respective roles in the regulation of luteolysis and the maternal recognition of pregnancy. In Expt 1, uterine biopsies were collected from four cows throughout three oestrous cycles each, to provide daily samples. In Expt 2, uterine tissue was collected on days 12, 14, 16 and 18 of the oestrous cycle (n = 20) or early pregnancy (n = 16). Oxytocin receptor, oestrogen receptor alpha and progesterone receptor mRNAs were localized by in situ hybridization, and localization of oestrogen receptor and progesterone receptor was confirmed by immunocytochemistry. All three receptors showed time- and cell-specific expression patterns. Oestrogen receptor alpha increased in all regions at oestrus but high concentrations were also found in the luminal epithelium during the mid-luteal phase and in the deep glands throughout the oestrous cycle. Progesterone receptor expression was higher in the stroma than it was in the types of epithelial cell, and increased expression was observed at oestrus and during the early luteal phase. The cyclical upregulation of oxytocin receptors in the luminal epithelium on about day 16 was not related to preceding changes in the endometrial expression of either oestradiol alpha or progesterone receptors. During early pregnancy, oxytocin receptor expression was suppressed. Oestrogen receptor a concentrations increased in the non-pregnant cows and decreased in the pregnant cows between days 16 and 18, but these changes followed rather than preceded the upregulation of oxytocin receptors in the non-pregnant cows. It is concluded that the initial upregulation of oxytocin receptors in the luminal epithelium, which triggers luteolysis, is not associated directly with changes in expression of oestrogen receptor alpha.

Immune cells in the corpus luteum: friends or foes?

Abstract: 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 2α , 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.

Effects of feeding tuna oil on the lipid composition of pig spermatozoa and in vitro characteristics of semen

Abstract: The aim of the present study was to characterize the effects of feeding tuna oil on the lipid and fatty acid composition of boar spermatozoa and to relate changes in composition to boar semen characteristics. Ten boars were paired by age and allocated to one of two diets (five boars per diet). The diets, which were offered for 6 weeks, consisted of a basal diet that was either unsupplemented or supplemented with 30 g tuna oil kg(-1) diet. Adding tuna oil to the diet increased the ether extract concentration of the diets fed from 65 to 92 g kg(-1) dry matter and supplied 10.5 g long chain polyunsaturated (n-3) fatty acids per 100 g total fatty acids. There were no changes in semen fatty acid composition after 3 weeks of feeding tuna oil. However, after 5 and 6 weeks, the proportions (g per 100 g total fatty acids) of 22:6(n-3) in sperm phospholipid fatty acids were increased from 34.5 to 42.9 g by feeding tuna oil and 22:5(n-6) decreased from 29.8 to 17.9 g. No changes were observed in other sperm lipids or seminal plasma phospholipids as a result of the diets fed. Feeding tuna oil increased the proportion of spermatozoa with progressive motility and with a normal acrosome score and reduced the proportion of spermatozoa with abnormal morphologies.

Role of gonadotrophins in regulating numbers of Leydig and Sertoli cells during fetal and postnatal development in mice.

Abstract: The role of the gonadotrophins in regulating numbers of Leydig and Sertoli cells during fetal and postnatal development was examined using normal mice and hypogonadal (hpg) mice, which lack circulating gonadotrophins. The disector method was used to determine the number of cells from day 16 of gestation until adulthood. The numbers of Leydig cells did not change significantly between day 16 of gestation and day 5 after parturition in normal mice and were not significantly different from numbers in hpg mice at any age up to day 5 after parturition. There was a 16-fold increase in the number of Leydig cells in normal mice between day 5 and day 20 after parturition, followed by a further doubling of number of cells between day 20 and adulthood. The number of Leydig cells in hpg testes did not change between day 5 and day 20 after parturition but doubled between day 20 and adulthood so that the number of cells was about 10% of normal values from day 20 onwards. Leydig cell volume was constant in normal animals from birth up to day 20 and then showed a 2.5-fold increase in adult animals. Leydig cell volume was normal in hpg testes at birth but decreased thereafter and was about 20% of normal volume in adult mice. The number of Sertoli cells increased continuously from day 16 of gestation to day 20 after gestation in normal mice and then remained static until adulthood. The number of Sertoli cells in hpg testes was normal throughout fetal life but was reduced by about 30% on day 1 (day of parturition). Thereafter, Sertoli cells proliferated at a slower rate but over a longer period in the hpg testis so that on day 20 after parturition the number of Sertoli cells was about 50% of normal values, whereas in adult mice the number was 65% of normal. The number of gonocytes did not change between day 16 of gestation and day 1 and did not differ between normal and hpg testes. The number of gonocytes increased nine-fold in normal testes but only three-fold in hpg testes between day 1 and day 5 after parturition. Gonocytes differentiated into spermatogonia in both normal and hpg testes between day 5 and day 20 after parturition. These results show: (i) that fetal development of both Sertoli and Leydig cells is independent of gonadotrophins; (ii) that normal differentiation and proliferation of the adult Leydig cell population (starting about day 10 after parturition) is dependent on the presence of gonadotrophins; and (iii) that the number of Sertoli cells after birth is regulated by gonadotrophins, although proliferation will continue, at a lower rate and for longer, in the absence of gonadotrophins.

Micronutrient programming of development throughout gestation

Abstract: 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.

Corticotrophin-releasing hormone and human parturition.

Abstract: Corticotrophin-releasing hormone (CRH), the hypothalamic peptide that controls function of the pituitary-adrenal axis in response to stress, is expressed in abundance in the human placenta and is present in high concentrations in maternal and fetal plasma during late pregnancy. A number of lines of evidence now imply a role for this hormone in the control of parturition and fetal maturation in humans. It has been proposed that CRH, through interactions with oestrogen, adrenal steroids, prostaglandins and oxytocin, establishes positive-feedback loops that initiate parturition and labour. Excessive production of placental CRH has also been linked to preterm labour. However, there are a number of significant gaps in our knowledge of the function of this peptide in pregnancy. This review examines current evidence regarding the putative role of CRH in human parturition.

Intraovarian actions of oestrogen

Abstract: 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.

Trophoblast interferon and pregnancy.

Abstract: The maternal recognition of pregnancy in ruminants requires the production of interferons by the preimplantation blastocyst. These proteins, the trophoblast interferons (IFN-tau), are the products of a number of similar genes, the expression of which is controlled by characteristic promoter regions. They are expressed for a short period in high concentrations, and have antiluteolytic, antiviral, antiproliferative and immunomodulatory effects, through receptors on the endometrial epithelium. The antiluteolytic effects of IFN-tau result from inhibition of endometrial expression of the oxytocin receptor, through which circulating oxytocin stimulates episodic prostaglandin F2a production. Some of the properties of IFN-tau differ from those of other type I interferons, and they may have novel therapeutic effects. Because of their central role in early gestation, these proteins have excited the interest of reproductive physiologists. However, their other properties, and the fact that their expression is controlled so precisely, have made them of interest to a wide range of biologists.