bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of dietary-induced increases in circulating insulin concentrations during the early postpartum period on reproductive function in dairy cows
Authors: Gong JG , Lee WJ , Garnsworthy PC , Webb R
Published: March 2004

Although it has become increasingly clear that fertility in modern dairy cattle is declining in association with increased milk yields, the underlying mechanism is poorly understood. The first ovulation post partum is delayed in dairy cows undergoing selection for genetic merit for milk yield in association with lower circulating insulin concentrations. The aim of this study was to investigate whether feeding a diet to increase circulating insulin concentrations can overcome this delay in the first ovulation post partum. The experiment was a 2 x 2 factorial design (n = 10 per group) involving diet and genetic merit for milk yield. The dietary treatment started on the day of calving and lasted for 50 days. Plasma samples were collected each day and ovarian ultra-sonography was performed three times a week during the experimental feeding period. Milk yield was recorded each day, and body weight and body condition score were determined each week. Milk samples were collected three times a week from day 50 to day 105 post partum, and reproductive performance data were recorded for all the cows as part of the routine farm practice. The dietary treatment induced significant differences in plasma insulin concentrations in both high and low genetic merit cows. Although high genetic merit cows produced more milk, lost more body weight and had lower body condition scores during the experiment, no significant effect of diet was observed on these measurements. The high insulin inducing diet increased the proportion of cows ovulating within 50 days of calving and reduced the intervals from calving to first ovulation, and tended to reduce the intervals from calving to first service and to conception. These fertility parameters were also more favourable in low than in high genetic merit cows, but no interaction between diet and genetic merit was observed for any of these parameters. Genetic merit, but not diet, also affected the number of services required per conception and the conception rate. In conclusion, these results have confirmed that genetic selection for high milk yield is associated with a decrease in reproductive performance in dairy cows. More importantly, this study has demonstrated that it is possible to alleviate this problem by nutritional manipulation.
Behaviour of a sperm surface transmembrane glycoprotein basigin during epididymal maturation and its role in fertilization in mice
Authors: Saxena DK , Oh-Oka T , Kadomatsu K , Muramatsu T , Toshimori K
Published: March 2004

Basigin (bsg) is a transmembrane glycoprotein belonging to an immunoglobulin superfamily and is localized on the surface of the sperm tail. The behaviour of bsg during epididymal maturation and its role in fertilization were examined using an anti-bsg antibody. Spermatozoa from caput, corpus and cauda epididymides were immunostained by indirect immunofluorescence (IIF). Immunostaining revealed that bsg is localized on the principal piece of caput spermatozoa and the molecule was found on the middle piece during transit in the corpus and cauda epididymides. Concomitantly, the molecular mass of bsg was reduced from 37 kDa (testis) to 26 kDa (cauda epididymidis). IVF experiments were designed to assess the effect of anti-bsg antibody on the fertilization events. Anti-bsg antibody significantly inhibited primary binding to the cumulus-invested oocytes with intact zonae pellucidae in a dose-dependent manner. Consequently, the fertilization rate of cumulus-invested oocytes with intact zonae pellucidae was also inhibited. The bsg molecule was also detected on the head of live capacitated spermatozoa by IIF under IVF conditions. These findings indicate that testicular bsg is a glycosylated protein that undergoes molecular processing and deglycosylation during its transit in the epididymis. The bsg molecule that was detected on the sperm head after capacitation may facilitate the primary binding or might be involved in distinct events required for primary binding of spermatozoa to the zona pellucida during capacitation and sperm-cumulus interaction.
Influence of maternal size on placental, fetal and postnatal growth in the horse. I. Development in utero
Authors: Allen WR , Wilsher S , Turnbull C , Stewart F , Ousey J , Rossdale PD , Fowden AL
Published: March 2004

The interacting influences of maternal size and fetal genotype on placental and fetal development in the mare were assessed by comparing conventional within-breed Thoroughbred (Tb-in-Tb, n = 7) and Pony (P-in-P, n = 7) control pregnancies established by artificial insemination (AI) with between-breed (Tb-in-P, n = 8; deprived in utero condition and P-in-Tb, n = 7; luxurious in utero condition) experimental pregnancies established by embryo transfer. All foals were born spontaneously and the mean (+/- SEM) duration of gestation in the two groups of control mares was significantly different (P < 0.001) at 325 +/- 3.0 days for the P-in-P pregnancies and 339 +/- 3.0 days for the Tb-in-Tb pregnancies, whereas the durations of gestation for the two experimental groups were very similar and midway between those of the control pregnancies at 332 +/- 2.8 days for the Tb-in-P and 331 +/- 2.7 days for the P-in-Tb. Mean (+/- SEM) foal birth weight and the mean (+/- SEM) values for the mass, gross area and volume of the allantochorion were all highest in the seven Tb-in-Tb pregnancies (53.1 +/- 2.6 kg, 3.8 +/- 0.3 kg, 12.9 +/- 0.3 x 10(3) cm(2), 3.5 +/- 0.2 l, respectively) and lowest in the seven P-in-P control pregnancies (24.0 +/- 1.3 kg, 1.7 +/- 0.1 kg, 8.3 +/- 0.3 x 10(3) cm(2), 1.8 +/- 0.1 l, respectively). These parameters were higher in the seven P-in-Tb pregnancies (37.9 +/- 2.1 kg, 2.7 +/- 0.1 kg, 10.1 +/- 0.5 x 10(3) cm(2), 2.5 +/- 0.1 l, respectively) than in the eight Tb-in-P (33.0 +/- 2.4 kg, 2.3 +/- 0.2 kg, 9.0 +/- 0.5 x 10(3) cm(2), 2.1 +/- 0.1 l) experimental pregnancies. Foal birth weight was positively correlated with the mass (r = 0.84, P < 0.001), gross area (r = 0.87, P < 0.001) and volume (r = 0.91, P < 0.001) of the allantochorion, and maternal weight was also positively correlated with both the mass and gross area of the allantochorion (r = 0.64 and 0.69, respectively; both P < 0.001). Application of stereology to multiple random biopsies recovered from each placenta produced mean values for the surface density of microcotyledons on the allantochorion (S(v)). Values were higher in Thoroughbred than in Pony mares regardless of the breed of fetus being carried. Multiplication of S(v) by the volume of the allantochorion to give the total microscopic area of fetomaternal contact at the placental interface was also positively correlated with foal birth weight (r = 0.84, P < 0.001). Foal birth weight was determined by the microscopic area of fetomaternal contact of the placenta and there were no differences in foal weight per m(2) of placenta regardless of fetal or maternal genomes. Thus, the results indicate that in equids, maternal size interacts with both the maternal and fetal genotypes to control the rate and extent of fetal growth by influencing the gross area of the diffuse allantochorion, and the density, complexity and depth of the microcotyledons on its surface.
Placental peptides as markers of gestational disease
Authors: Page NM , Kemp CF , Butlin DJ , Lowry PJ
Published: March 2004

The human placenta produces a wide range of important peptides, of which an intricate balance is required throughout pregnancy. In a gestational disease, this balance may be disturbed and the identification of such changes may be used to detect a particular pathology or to ascertain its severity. This review considers the role and association of various placental peptide markers associated with the major gestational diseases including intrauterine growth retardation, pre-term labour, pre-eclampsia, chromosomal disorders, gestational diabetes and trophoblastic disease. Potential markers that may prove more reliable and specific in their diagnostic value and that may be used for identifying patients at risk are also discussed. The importance of the new fields of genomics and proteomics in the future discovery of new peptide markers is illustrated.
Increased ovulation rate in gilts treated with dihydrotestosterone
Authors: Cardenas H , Herrick  , Pope WF
Published: March 2004

Treatment with testosterone increases ovulation rate in pigs. The present study was conducted to examine the effects of 5alpha-dihydrotestosterone (DHT), a non-aromatizable androgen receptor ligand, on ovulation rate and amounts of androgen receptor and FSH receptor mRNAs in postpubertal gilts. In Expt 1, ovulation rate in response to daily i.m. injections of 0, 6, 60 or 600 microg DHT kg(-1) body weight from day 13 of the oestrous cycle (day 0 = day 1 of oestrus) to the following oestrus increased with each dose of DHT (P < 0.05). The mean increase in number of corpora lutea ranged from approximately three to 17 over the three dosages of DHT. In Expt 2, gilts treated daily with 60 microg DHT kg(-1) body weight during the early follicular phase (from day 13 to day 16), coincident with follicular recruitment, or the late follicular phase (day 17 to oestrus), had higher (P < 0.05) rates of ovulation compared with gilts that received vehicle, and were not different from gilts treated with DHT from day 13 to oestrus. Percentage recovery of day 3 embryos was not altered when gilts were treated from day 13 to day 16 or from day 17 to oestrus; however, treatment of gilts with DHT from day 13 to oestrus decreased recovery of day 3 (Expt 1) or day 11 (Expt 2) conceptuses. Daily administration of 6 microg DHT kg(-1) body weight to gilts from day 13 of the oestrous cycle to the following oestrus (Expt 3) did not affect the relative amounts of androgen receptor mRNA, but increased (P < 0.05) the amounts of FSH receptor mRNA in preovulatory follicles as determined by RT-PCR. The results of these experiments indicate that androgens may regulate ovulation rate in gilts. One of the roles of androgens might be regulation of the amounts of FSH receptor mRNA in ovarian follicles.
Plasma FSH, inhibin A and inhibin isoforms containing pro- and -alphaC during winter anoestrus, spring transition and the breeding season in mares
Authors: Watson ED , Heald M , Tsigos A , Leask R , Steele M , Groome NP , Riley SC
Published: March 2004

Ten mares were studied from February (winter anoestrus) to their second ovulation in the breeding season to investigate the relationship between resumption of ovarian cyclicity in the spring and circulating concentrations of FSH, inhibin A and inhibin isoforms containing pro- and -alphaC immunoreactivity. An additional four mares were studied during one oestrous cycle. Growth and regression of ovarian follicles were monitored by transrectal ultrasonography. The frequency of blood sampling varied from three times a week to once a day, depending on the follicular activity present. Concentrations of FSH, oestradiol, inhibin A and pro- and -alphaC isoforms were low during deep winter anoestrus when minimal follicular activity was present in the ovaries. During spring transition, an increase in FSH concentration preceded the emergence of each follicular wave. Concentrations of inhibins were significantly higher (P < 0.05) during growth of anovulatory follicles in spring transition than during winter anoestrus. Plasma concentrations of oestradiol and inhibin A were significantly higher (P < 0.001, P < 0.05, respectively) during the growth of preovulatory follicles than during the growth of transitional anovulatory follicles, but concentrations of inhibin pro-alphaC isoforms did not differ between the two types of follicle. During the oestrous cycle, there was a significant inverse relationship (P < 0.001) between concentrations of FSH and the inhibins. Plasma inhibin pro-alphaC isoforms, but not inhibin A, reached a peak on the day of ovulation. The results strongly indicate that FSH regulates growth of spring anovulatory and preovulatory follicles. Inhibins are likely to contribute to negative feedback on the release of FSH from the pituitary gland both during the transitional period and the breeding season in mares.
Characterization of mouse spermatogonia by transmission electron microscopy
Authors: Chiarini-Garcia H , Russell LD
Published: March 2004

Characteristics of the various type A, intermediate (In) and B spermatogonia were determined in C57BL/6J mice using transmission electron microscopy. Spermatogonia were photographed at all stages of the cycle of the seminiferous epithelium. Over 450 images were taken. Spermatogonia could be classified into As, Apr, Aal, A1 cells, A2 cells, A3 cells, A4 cells, intermediate type and type B cells primarily on the basis of nuclear and nucleolar characteristics. The most primitive spermatogonia (As, Apr, Aal) had mottled chromatin; A1 cells contained homogeneously finely granular chromatin throughout the nucleus; A2, A3, A4 and intermediate type spermatogonia had progressively increasing amounts of chromatin encrusting the nuclear envelope; type B spermatogonia had less heterochromatin along the nuclear envelope, although the particles were more dense and rounded than in intermediate type spermatogonia. Mitochondrial size and position of Golgi complexes varied in different types of spermatogonia. These data show that types of spermatogonia can be differentiated such that these characteristics can be used in functional studies.
Differential effects of glucose and fructose on hexose metabolism in dog spermatozoa
Authors: Rigau T , Rivera M , Palomo MJ , Fernandez-Novell JM , Mogas T , Ballester J , Pena A , Otaegui PJ , Guinovart JJ , Rodriguez-Gil JE
Published: March 2004

Incubation of dog spermatozoa with 10 mmol l(-1) glucose or fructose rapidly increased the intracellular content of glucose 6-phosphate and fructose 6-phosphate, although the effect of fructose was greater. These effects were correlated with increases in ATP, ribose 5-phosphate and glycogen contents, and in the rates of formation of L-lactate and CO2. In all cases, except for ATP and glycogen, the effect of fructose was greater than that of glucose. The total hexokinase activity of the crude extracts of dog spermatozoa was more sensitive to fructose than to glucose at lower concentrations (0.1-3.0 mmol l(-1)). Both monosaccharides induced a fast and intense increase in the overall tyrosine phosphorylation of dog spermatozoa, although their specific induced-phosphorylation patterns differed slightly. Glut 3 and Glut 5 hexose transporters were the main hexose transporters in dog spermatozoa; however, other possible SGLT family-related hexose transporters were also localized. These data indicate that, at concentrations from 1 mmol l(-1) to 10 mmol l(-1), fructose has a stronger effect than glucose on hexose metabolism of dog spermatozoa. These differences appear to be related to variations in the sensitivity of hexokinase activity. Moreover, the differential hexose metabolism induced by the two sugars had distinct effects on the function of dog spermatozoa, as revealed by the diverse patterns of tyrosine phosphorylation.
Gap junctions and ovarian folliculogenesis
Authors: Kidder GM , Mhawi AA
Published: March 2004

Gap junctions are collections of intercellular membrane channels that allow adjacent cells to share small molecules (< 1 kDa). Gap junction channels are composed of connexins, a homologous family of more than 20 proteins. In developing follicles, gap junctions couple the growing oocyte and its surrounding follicle cells into a functional syncytium. This review summarizes evidence on the expression of various connexins in developing follicles and the likely roles that some of the connexins play, on the basis of findings from gene targeting experiments in mice. Gap junctions between cumulus cells contain predominantly connexin43, and this connexin has also been detected using immunoelectron microscopy in a small minority of gap junctions at the oocyte surface. The importance of connexin43 for granulosa cell function is demonstrated by the fact that follicles lacking this connexin arrest in early preantral stages and produce incompetent oocytes. Connexin37 appears to be the only connexin contributed by oocytes to the gap junctions coupling them with granulosa cells, and loss of this connexin interferes with the development of antral follicles. The expression of multiple connexins in developing follicles is thus likely to reflect the multiple functions served by gap junctional communication in folliculogenesis.
Progesterone stimulation of fluid absorption by the rat uterine gland
Authors: Naftalin RJ , Thiagarajah  , Pedley KC , Pocock VJ , Milligan SR
Published: March 2004

The purpose of this study was to test the hypothesis that the steroid environment affects fluid absorption by the uterine glands. Laser scanning confocal microscopy of the distribution of an extracellular marker (fluorescein isothiocyanate-labelled dextran) within rat uterine glands showed that the endometrial glands change their fluid handling characteristics under different hormonal conditions. Under progesterone dominance, the glands showed an amiloride-sensitive dextran accumulation indicating sodium-dependent fluid absorption; however, this was absent in the oestrogen-dominated state. The rate of fluid uptake in the progesterone-stimulated gland opening was estimated to be approximately 1 x 10(-4) cm s(-1), requiring a suction pressure of between 10 and 20 mm Hg at the mucosal surface. This study provides the first direct evidence of fluid absorption by the uterine glands. Such absorption may provide the mechanism for closure of the uterine lumen and immobilization of the blastocyst necessary for implantation.
Morphological evidence for uncontrolled proteolytic activity during the ovulatory process in indomethacin-treated rats
Authors: Gaytan F , Tarradas E , Morales C , Bellido C , Sanchez-Criado JE
Published: March 2004

The ovulatory process in cyclic rats was studied after prostanoid synthesis was blocked using indomethacin. Animals were injected at 12:00 h in pro-oestrus with 1.0 mg indomethacin or vehicle (olive oil) and killed at 18:30 h in pro-oestrus, at 02:00, 09:00 and 19:00 h in oestrus and at 09:00 h in metoestrus. Additional rats injected with 0.5 or 4.0 mg indomethacin were killed at 09:00 h in oestrus. No differences in either morphology or serum LH concentrations were found between vehicle or indomethacin-treated rats at 18:30 h in pro-oestrus. However, from 02:00 h in oestrus onward, the process of follicle rupture was altered considerably in indomethacin-treated rats, irrespective of the dose. Early vascular changes, observed in control rats at the apex of the follicle, were absent in indomethacin-treated rats. In some follicles, disruption of the theca layers, invasion of the perifollicular tissue by granulosa cells and follicular fluid, and release of the oocyte to the ovarian interstitium were observed at 02:00 h in oestrus. A small number of follicles ruptured at the ovarian surface. Furthermore, invasion of interstitial tissue, rupture of blood vessel walls, production of emboli of granulosa cells and follicular fluid, and inflammatory reactions were observed in oestrus and metoestrus. The results of the present study demonstrate uncontrolled proteolytic activity, and indicate that abnormal follicle rupture (but not inhibition of follicle rupture) is responsible for ovulation failure in indomethacin-treated rats.
Regulation of cyclooxygenase activity and progesterone production in the rat corpus luteum by inducible nitric oxide synthase
Authors: Hurwitz A , Finci-Yeheskel Z , Milwidsky A , Mayer M
Published: March 2004

This study explores interactions between the nitric oxide synthase (NOS) and the cyclooxygenase (COX) pathways in the regulation of progesterone production in early corpus luteum cells of rats. Nitric oxide (NO), prostaglandin E (PGE) and progesterone production was analysed in luteal cells of the rat corpus luteum exposed to inhibitors of non-specific NOS, inhibitors of inducible NOS (iNOS) and inhibitors of COX. Equine chorionic gonadotrophin (eCG)/hCG-primed rat corpus luteum cells produced NO, PGE and progesterone in a linear manner during 66 h of culture. Exposure of the cells to the non-specific NOS inhibitor, N(omega)-nitro-L-arginine (0.15 mmol l(-1)) for 48 h reduced NO, PGE and progesterone production to 21, 32 and 60% of that of the controls, respectively (P < 0.05 to P < 0.01). Another non-specific NOS inhibitor, N(omega)-methyl-L-arginine, produced similar inhibitions. Exposure of the cultured cells to S-ethylisothiourea (1 mmol l(-1)), a selective inhibitor of iNOS, suppressed the production of NO by 63%, PGE by 69% and progesterone by 48%. These findings indicate that production of PGE is regulated partly by iNOS, and that progesterone is probably regulated indirectly by the secondary changes in PGE. The addition of arachidonic acid to N(omega)-methyl-L-arginine-treated cells resulted in a significant increase in PGE and progesterone production (273 and 186%, respectively) without stimulating NO production. In contrast to the regulation exerted by the NO system on COX activity, the COX system does not modulate NO production in this model. This notion stems from the observation that the COX inhibitors acetylsalicylic acid (5 mmol l(-1)) and indomethacin (5 micromol l(-1)) suppressed PGE by 86 and 89%, respectively, and progesterone by 34 and 57%, respectively, but failed to inhibit NO production. The results from the present study indicate that iNOS-mediated NO production is involved in stimulating PGE synthesis in rat luteal cells, which may upregulate progesterone production.
Variations in modifications of sugar residues in hamster zona pellucida after in vivo fertilization and in vitro egg activation
Authors: El-Mestrah M , Kan FW
Published: March 2004

Lectins were used as probes in conjunction with quantitative analysis to investigate the distribution of different carbohydrate residues in hamster zona pellucida and their possible modification patterns after in vivo fertilization and in vitro egg activation. Several lectins including HPA, WGA, RCA-I, PNA, DSA, BSAIB(4), DBA, AAA and MAA were used to label the zona pellucida of both unfertilized and fertilized eggs. With the exception of PNA and BSAIB(4), the same lectins were also used to label the zona pellucida of oocytes activated in vitro. A multicomparison quantitative analysis of the density of labelling in the inner and outer regions of the zona pellucida before and after fertilization in vivo, as well as after in vitro egg activation, was performed. Of all the lectins studied, preferential localization of labelling by RCA-I and DSA to the inner zona pellucida of unfertilized eggs was observed. After in vivo fertilization, there was an increase in labelling in the inner region of the zona pellucida when thin sections of fertilized oocytes were incubated with HPA, BSAIB(4) and AAA. Although increased labelling by RCA-I was observed, a significant decrease in labelling intensity was obtained with WGA and the sequence Neu-WGA in both the inner and outer zona pellucida of fertilized oocytes. A significant increase in the density of labelling with WGA was also observed after digestion with neuraminidase. In parallel, when hamster oocytes activated in vitro were compared with those fertilized in vivo, a difference in lectin-gold labelling was observed in both the inner and outer region of the zona pellucida. Labelling with HPA, WGA, DSA and MAA increased significantly in both the inner and outer regions of the zona pellucida, whereas labelling by DBA significantly decreased in the inner portion of the zona pellucida. After neuraminidase treatment, a significant increase in labelling density was observed when thin sections of in vitro-activated oocytes were incubated with WGA. These results demonstrate: (i) the post-fertilization modifications of major saccharidic determinants that may play a role in the sperm-egg interaction process of fertilization in vivo; and (ii) that the modified properties of zonae pellucidae of fertilized and in vitro-activated eggs resulting from the action of hydrolytic enzymes, as well as glycoproteins released through exocytosis of cortical granules, are not identical.
Role of myometrial activity in sperm transport through the genital tract and in fertilization in sows
Authors: Langendijk P , Bouwman EG , Kidson A , Kirkwood RN , Soede NM , Kemp B
Published: March 2004

The effects of stimulation and suppression of uterine contractility at about the time of insemination on sperm distribution and fertilization in multiparous sows are described. For assessment of fertilization, sows were inseminated about 28 h before (synchronized) ovulation and killed at day 5 after ovulation (n = 53). For assessment of sperm distribution, sows were inseminated about 20 h before expected ovulation and were killed 12 h later (n = 26). At 10 min before insemination, sows received an intrauterine infusion of one of three solutions: (i) saline (control); (ii) 0.60 mg clenbuterol hydrochloride to suppress contractility; or (iii) 1 mg cloprostenol to stimulate contractility. Both clenbuterol and cloprostenol reduced median fertilization rate (P < 0.05) and median number of accessory sperm cells (P < 0.05). Distribution of sperm cells was also affected by treatments. Clenbuterol increased, and cloprostenol decreased, the number of sperm cells (P < 0.05) in the proximal 20 cm of the uterine horn and in the uterotubal junction. In addition, clenbuterol tended to increase and cloprostenol tended to decrease the number of sperm cells in the isthmus, although these effects were not significant. However, relative to the number of sperm cells in the uterus, clenbuterol treatment reduced the number of sperm cells in the uterotubal junction and oviduct, in contrast to cloprostenol. Cloprostenol increased the reflux of semen during insemination. It is hypothesized that suppression of uterine contractility increases transuterine transport time, reducing the ability of sperm cells to enter the uterotubal junction and the oviduct. Stimulation of uterine contractility above a certain level probably increases reflux and impedes transuterine transport of sufficient numbers of sperm cells.
Gonadotrophin surge-induced upregulation of mRNA for plasminogen activator inhibitors 1 and 2 within bovine periovulatory follicular and luteal tissue
Authors: Dow MP , Bakke LJ , Cassar CA , Peters MW , Pursley  , Smith GW
Published: March 2004

The serine proteinases, tissue-type (tPA) and urokinase (uPA) plasminogen activator, are implicated in the ovulatory processes via their ability to convert plasminogen to its active form, plasmin. One mechanism for regulation of plasmin-directed ovarian extracellular matrix remodelling during follicle rupture and corpus luteum formation is through inhibition of plasminogen activation by the plasminogen activator inhibitors (PAI-1 and PAI-2). The effect of the preovulatory gonadotrophin surge on the temporal and spatial regulation of expression of PAI-1 and PAI-2 mRNA and PAI activity in preovulatory bovine follicles and new corpora lutea collected at 0, 6, 12, 18, 24 and 48 h after a GnRH-induced gonadotrophin surge was examined. Both PAI-1 and PAI-2 mRNAs were upregulated markedly after the gonadotrophin surge, with the highest expression observed in follicles collected at about the time of ovulation (24 h) and in corpora lutea (48 h). PAI-1 mRNA was localized primarily to the thecal layer of preovulatory follicles. In contrast, PAI-2 mRNA was localized specifically to the granulosa cell layer. Significant PAI activity was detected in follicle extracts, but temporal or spatial differences in PAI activity were not detected in response to the gonadotrophin surge. These results indicate that PAI-1 and PAI-2 mRNAs are upregulated in preovulatory bovine follicles after the gonadotrophin surge in a cell-specific way. Regulation of PAI-1 and PAI-2 may help to control plasminogen activator activity associated with ovulation and early corpus luteum formation.
Differentiation of chicken gonad as an endocrine organ: expression of LH receptor, FSH receptor, cytochrome P450c17 and aromatase genes
Authors: Akazome Y , Abe T , Mori T
Published: March 2004

The gonad is an endocrine organ secreting sex hormones and also a target of pituitary gonadotrophins. The expression of mRNAs encoding LH receptor (LHR), FSH receptor (FSHR), P450c17 and P450aromatase in the developing gonads of embryos between day 4 and day 6 of incubation was determined using a RT-PCR to elucidate the chicken gonad as a target organ of gonadotrophins. Although expression of mRNAs encoding LHR, FSHR and P450c17 was detected at day 4 of incubation in both sexes, mRNA encoding P450aromatase appeared at day 6 in female embryos only, indicating that mRNAs encoding gonadotrophin receptors can be identified before sexual differentiation. Quantitative PCR analysis revealed that expression of mRNA encoding LHR and FSHR remained low in male gonads from day 4 to day 6 of incubation, whereas they increased on day 6 in female gonads. The sexual dimorphism in the expression of mRNAs encoding LHR and FSHR was confirmed in the sexually differentiated gonads of embryos at day 12 of incubation (LHR in ovary ratio LHR in testis = 7 ratio 1; FSHR in ovary ratio FSHR in testis = 9 ratio 1).
Relationship between seasonal changes in spermatogenesis in the juvenile ostrich (Stuthio camelus) and the presence of the LH receptor and 3beta-hydroxysteroid dehydrogenase
Authors: Madekurozwa MC , Chabvepi TS , Matema S , Teerds KJ
Published: March 2004

The immunohistochemical localization of the LH receptor and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) was studied in the testis of the juvenile ostrich (Stuthio camelus) throughout a 1 year period. Spermatogenic activity of juvenile birds changed throughout the year, as has been reported previously for sexually mature birds. During the active stage of the testicular cycle, from September to January, spermatogenesis progressed up to the stage of formation of spermatozoa, although spermatozoa could not be detected in the epididymis. Leydig cells stained intensely with antibodies against the LH receptor and 3beta-HSD during the quiescent, recrudescent and active phases of the testicular cycle. During the regressive phase, there was a slight decrease in immunostaining for 3beta-HSD in these cells. These results indicate that Leydig cells in the testis of the juvenile ostrich are able to respond to LH and are capable of steroid synthesis. Furthermore, in juvenile (prepubertal) ostriches, spermatogeneic activity can be observed and, as in mature birds, spermatogenesis undergoes seasonal changes.
Segregation of mitochondrial DNA (mtDNA) in human oocytes and in animal models of mtDNA disease: clinical implications
Authors: Poulton J , Marchington DR
Published: March 2004

Mitochondrial DNA (mtDNA) is almost entirely maternally inherited. Thousands of copies of mtDNA are present in every nucleated cell and in most normal individuals these are virtually identical (homoplasmy). mtDNA diseases may be caused by mutations in either mitochondrial or nuclear genes and, hence, give rise to maternal or autosomal patterns of inheritance. Antenatal diagnosis of mitochondrial diseases based on chorionic villous sampling is available for Mendelian disorders and the syndromes caused by mutations at bp 8993 (associated with Leigh's syndrome and neurogenic weakness, ataxia and retinitis pigmentosa (NARP)). However, prenatal diagnosis of many other maternally inherited mtDNA diseases is less reliable because it is not possible to predict with confidence the way in which heteroplasmic mtDNA mutations segregate within tissues and find clinical expression. This review focuses on the substantial progress in genetics that has been made recently, and on the management options that clinicians can offer to families.
Detection and location of the enzymes of de novo pyrimidine biosynthesis in mammalian spermatozoa
Authors: Carrey EA , Dietz C , Glubb DM , Loffler M , Lucocq JM , Watson PF
Published: March 2004

Enzymes of the pathway for de novo biosynthesis of pyrimidine nucleotides have been reported in spermatozoa from fruitfly and mammals. The aim of the present study was to test the hypothesis that the enzymes for biosynthesis of uridine monophosphate (UMP) are concentrated near the mitochondria, which are segregated in the mid-piece of spermatozoa. Baby hamster kidney fibroblasts were compared with spermatozoa from rams, boars, bulls and men. Antibodies raised against synthetic peptides from sequences of the multienzyme polypeptides containing glutamine-dependent carbamyl phosphate synthetase, aspartate transcarbamylase and dihydroorotase (CAD) and UMP synthase, which catalyse reactions 1-3 and 5-6, respectively, were used, together with an affinity-purified antibody raised against dihydroorotate dehydrogenase (DHODH), the mitochondrial enzyme for step 4. Western blot analysis, immunofluorescent microscopy and immunoelectron microscopy confirmed that CAD and UMP synthase are found in the cytoplasm around and outside the mitochondria; DHODH is found exclusively inside the mitochondria. CAD was also located in the nucleus, where it has been reported in the nuclear matrix, and in the cytoplasm, apparently associated with the cytoskeleton. It is possible that CAD in the cytoplasm has a role unconnected with pyrimidine biosynthesis.
Impact of maternal nutrition during pregnancy on pituitary gonadotrophin gene expression and ovarian development in growth-restricted and normally grown late gestation sheep fetuses
Authors: Da Silva P , Aitken RP , Rhind SM , Racey PA , Wallace JM
Published: March 2004

The influence of maternal nutrition during pregnancy on anterior pituitary gonadotrophin gene expression and ovarian development in sheep fetuses during late gestation was investigated. Embryos recovered from superovulated adult ewes that had been inseminated by a single sire were transferred, singly, into the uteri of adolescent recipients. After embryo transfer, adolescent ewes were offered a high or moderate amount of a complete diet. Pregnancies were terminated at day 131 +/- 0.6 of gestation and the fetal brain, anterior pituitary gland and gonads were collected. Gonadotrophin gene expression (LHbeta and FSHbeta subunits) in the fetal pituitary gland was examined using in situ hybridization. Ovarian follicular development was quantified in haematoxylin- and eosin-stained ovarian sections embedded in paraffin wax. Six dams that were offered a high nutrient intake carried normal-sized fetuses (weight within +/- 2 SD of mean weight for control fetuses from dams fed a moderate level of complete diet) and 13 dams carried growth-restricted fetuses (weight +/- 2 SD of mean weight for control fetuses from dams fed a moderate level of complete diet). Mean placental masses in these groups were 354 +/- 24.5 and 230 +/- 21.1 g, respectively, compared with 442 +/- 54.3 g in the dams that were offered a moderate nutrient intake (n = 6). Growth-restricted fetuses from dams offered a high nutrient intake showed higher pituitary LHbeta mRNA expression (P < 0.05) than normal-sized fetuses from dams offered a moderate nutrient intake (252 +/- 21.6 and 172 +/- 23.6 nCi g(-1), respectively). FSHbeta mRNA expression was not influenced by growth status. Fewer follicles (primarily in the resting pool) were observed in the ovaries of both growth-restricted (P < 0.002) and normal-sized fetuses from dams offered a high nutrient intake (P < 0.01) compared with normal-sized fetuses from dams offered a moderate nutrient intake. Irrespective of nutritional treatment, the total number of follicles was positively associated with placental mass (P < 0.01). Thus, a high maternal nutrient intake during adolescent pregnancy had a negative influence on ovarian follicular development in fetuses as determined during late gestation.
Differences in splicing of mRNA encoding LH receptor in theca cells according to breeding season in ewes
Authors: Abdennebi L , Lesport AS , Remy JJ , Grebert D , Pisselet C , Monniaux D , Salesse R
Published: March 2004

Splice variants of mRNA encoding the LH receptor (LHR) during follicular development were characterized in cyclic and non-cyclic ewes. Granulosa and theca cells were collected from individual follicles. After amplification by RT-PCR of a region situated between exon 9 and exon 11 of the LHR gene, three distinct bands, LHR1 (full length), LHR2 (deletion of exon 10), LHR3 (deletion of 262 bp in exon 11), were observed in the granulosa and theca cells of ovine antral follicles of various sizes (2.5-6.0 mm). Expression of LHR mRNA in theca cells varied with the annual cycle of reproduction (P < 0.001), and was highly expressed in all classes of follicle collected from anoestrous ewes (1.3 +/- 0.1, n = 8 in small follicles; 1.8 +/- 0.2, n = 8 in medium follicles; 1.7 +/- 0.3, n = 4 in large follicles; arbitrary units) compared with follicles collected from oestrous ewes (0.19 +/- 0.06, n = 8 in small follicles; 0.2 +/- 0.04, n = 9 in medium follicles; 0.18 +/- 0.04, n = 5 in large follicles). During the breeding season, no differences in the relative expression of the different splice variants were observed according to follicle size. In contrast, during anoestrus, LHR3 mRNA was significantly more abundant in large (6.0-6.5 mm) and medium (4.0-5.5 mm) than it was in small (2.5-3.5 mm) follicles. These results indicate that RNA alternative splicing plays a role in the seasonal and physiological control of LH receptor expression in theca cells.
Production of inhibin A not B in rams: changes in plasma inhibin A during testis growth, and expression of inhibin/activin subunit mRNA and protein in adult testis
Authors: McNeilly AS , Souza CJ , Baird DT , Swanston IA , McVerry J , Crawford J , Cranfield M , Lincoln GA
Published: March 2004

Previous studies have shown that changes in the plasma concentrations of immunoreactive inhibin measured by radioimmunoassay occur in parallel with growth and regression of the testes during a reproductive cycle in adult Soay rams induced by exposure to an artificial lighting regimen of alternating 16 week periods of long days and short days. With the development of new two-site ELISAs for sheep inhibin A and inhibin B, we have re-examined the relationship between FSH and dimeric, biologically active inhibin in the reproductive cycle in adult Soay rams. No signal was generated by sheep testicular extract, ram or ewe plasma, or sheep ovarian follicular fluid in the inhibin B ELISA. In contrast, ram plasma contained significant activity in the inhibin A ELISA, which diluted in parallel to the inhibin A standard, and was abolished by preincubation of ram plasma with monoclonal antibodies specific for the betaA, but not the betaB, subunit. These results indicate that the ram is the first adult male mammalian species identified to date in which the testes produce and secrete dimeric inhibin A and not inhibin B. Northern blot analysis and immunocytochemistry confirmed the presence of alpha, betaA and betaB inhibin/activin subunit mRNA and protein in the testes of adult rams. Changes in plasma inhibin A concentrations occurred in parallel with the growth and regression of the testes during the long day: short day: long day lighting regimen in adult Soay rams, confirming our previous observations with immunoreactive inhibin. During the growth phase of the testes in the first 8 weeks of exposure to short days there was a positive correlation between plasma FSH and inhibin A concentrations, indicating that during this phase the secretion of inhibin A is stimulated by FSH and that inhibin A did not act as a negative feedback hormone on FSH secretion. From week 8.5 to week 16.0 of exposure to short days, there was a negative correlation between FSH and testosterone concentrations, but not inhibin, indicating that when inhibin concentrations are high, testosterone acts as the negative regulator of FSH secretion. Thus, in intact adult rams, when the testes are fully active it appears that inhibin A may sensitize the pituitary to the negative feedback effects of testosterone, at which time they act synergistically to maintain plasma concentrations of FSH.
Evaluation of the immunocontraceptive potential of Escherichia coli-expressed recombinant dog ZP2 and ZP3 in a homologous animal model
Authors: Srivastava N , Santhanam R , Sheela P , Mukund S , Thakral SS , Malik BS , Gupta SK
Published: March 2004

Dog zona pellucida glycoprotein 2 (dZP2), excluding the N-terminal signal sequence and the C-terminal transmembrane-like domain, was cloned and expressed as a polyhistidine fusion protein in Escherichia coli to evaluate the immunocontraceptive efficacy of ZP glycoproteins. The recombinant dZP2 (rec-dZP2) revealed a 70 kDa band corresponding to the full length transcript, as well as several low molecular mass fragments in western blot analysis. In addition to rec-dZP2, E. coli expressed recombinant dog ZP glycoprotein 3 (rec-dZP3), which has also been evaluated for its efficacy to block fertility in a homologous system. Three groups of female dogs (n = 4 per group) were immunized with rec-dZP2 conjugated to diphtheria toxoid (rec-dZP2-DT), rec-dZP3 conjugated to DT (rec-dZP3-DT) and DT alone. Immunization of female dogs with rec-dZP2-DT and rec-dZP3-DT led to generation of antibodies against the respective ZP proteins as well as to DT. Subsequent to mating, the four female dogs immunized with rec-dZP2-DT all conceived, which is indicative of failure of the anti-rec-dZP2 antibodies to block fertility. In the group of dogs immunized with rec-dZP3-DT, three of four animals did not conceive when mated with males of proven fertility. The block in fertility was associated with anti-dZP3 antibody titres. Ovarian histopathology revealed that the block in fertility in the group immunized with rec-dZP3-DT is probably manifested by inhibition in the development of follicles and is due to atretic changes in the zona pellucida. These results, although preliminary, indicate that immunization with dZP3 may be a feasible proposition to control dog populations provided that adequate antibody titres are achieved.
Expression of the insulin-like growth factor (IGF) system in the bovine oviduct at oestrus and during early pregnancy
Authors: Pushpakumara PG , Robinson RS , Demmers KJ , Mann GE , Sinclair KD , Webb R , Wathes DC
Published: March 2004

Early mammalian embryo development in vitro can be enhanced by co-culture with oviductal cells and by the addition of insulin-like growth factors (IGFs). This study examined the expression patterns of the oviductal IGF system in cattle in relation to the number of days after oestrus and the presence or absence of embryos. Oviducts were collected from: (i) 66 nulliparous heifers on day 3, day 6 or day 16 after insemination and from (ii) ten non-pregnant, lactating cows on day 0 or day 1 of the oestrous cycle. Oviducts were coiled, frozen whole and sectioned for in situ hybridization. Expression patterns of mRNAs encoding IGF-I, IGF-II, type 1 IGF receptor (IGF-1R), and the IFG binding proteins (IGFBP)-1, -3 and -5 were determined from autoradiographs. Separate measurements were made for the mucosa and muscle layers of the infundibulum, ampulla and isthmus. None of the parameters measured differed between heifers with or without the presence of an embryo. mRNAs encoding IGF-I and IGF-1R were present in the mucosa and muscle of all three oviductal regions, and the highest value of IGF-I mRNA was measured in heifers on day 3. IGF-II mRNA was expressed predominantly in the muscle wall. IGFBP-1 mRNA was not detectable, whereas mRNAs encoding IGFBP-3 and -5 were expressed in both the muscle and mucosa. IGFBP-3 expression was higher in cows on day 0 and day 1 of the oestrous cycle than in heifers on day 3, day 6 and day 16 after insemination. A peak of IGFBP-5 expression was reached on day 6. Locally or systemically produced IGFs, regulated by IGFBPs, may act directly on the embryo or indirectly via modulation of oviductal secretions and muscular activity to influence the success of early embryo development.
Effect of dextrans on cryopreservation of goat cauda epididymal spermatozoa using a chemically defined medium
Authors: Kundu CN , Chakrabarty J , Dutta P , Bhattacharyya D , Ghosh A , Majumder GC
Published: March 2004

Experiments were carried out to investigate the cryoprotecting potential of dextrans (ranging from 10 to 2000 kDa) using a synthetic model system developed recently in this laboratory. Goat spermatozoa from the cauda epididymidis were extracted in a chemically defined medium (modified Ringer's solution) and assayed for motility using a phase-contrast microscope. The sperm cells were subjected to a freezing protocol in a computer-controlled biofreezer (cooling at 0.25 degrees C min(-1) to 5 degrees C; 5 degrees C min(-1) to -20 degrees C; 20 degrees C min(-1) to -100 degrees C) and plunged into liquid nitrogen. The frozen cells were thawed rapidly at 37 degrees C in a thermostatic waterbath. In the absence of dextran, all the spermatozoa lost their motility. The cryoprotecting efficacy of each dextran was found to be biphasic in nature. Initially, as the concentration of dextran was increased, the recovery of sperm motility also increased and reached an optimum value; however, with further increases in dextran concentration, the recovery of motility decreased sharply. Of all the sugar polymers tested, 10 kDa dextran showed the highest cryoprotecting efficacy, whereas the 2000 kDa sugar polymer was the least active. Dextrans of 10, 40, 73, 173, 252, 500 and 2000 kDa offered maximum cryorecovery of forward motility to the extent of approximately 23%, 21%, 19%, 18%, 16%, 15% and 8%, respectively. Optimum concentrations of these dextrans for cryoprotection of sperm motility were 8.42, 2.50, 1.09, 0.37, 0.31, 0.10 and 0.04 mmol l(-1), respectively. It thus appears that each dextran has a characteristic cryoprotection profile. The data show that the cryoprotecting efficacy and optimum cryoprotecting concentrations of dextrans are inversely related to their molecular mass. Dextran also served as a significant cryoprotectant in the presence of glycerol (0.87 mol l(-1)) and dimethyl sulphoxide (0.76 mol l(-1)), which are well known cryoprotectants; the action of the combined cryoprotectants was almost additive. The presence of glycerol or glycerol plus dimethyl sulphoxide caused a significant reduction (from 8.42 to 6.27 mmol l(-1)) in the optimum concentration of dextran. In the presence of the three cryoprotectants, recovery of sperm motility was as high as 58% (forward motility) and 60% (total motility).
Caspase-mediated apoptosis in the vertebrate ovary
Authors: Johnson AL , Bridgham JT
Published: March 2004

In the vertebrate ovary, apoptosis is the process by which excess or non-viable germ and granulosa cells are eliminated early in ontogeny (often beginning before birth), and thereafter continuously throughout reproductive life. Accordingly, an excessively high rate or abnormal triggering of such cell death (and, by implication, follicle atresia) can negatively affect fertility. Programmed cell death involves the integration of many pathways and intracellular proteins, and central among these at almost every stage are members of the caspase family. Relatively little attention has been focused upon the ovary with regards to elucidating initiator and effector members of the caspase family, and pathways by which they are activated and inactivated. The present review briefly describes vertebrate caspases and the regulation of their function in non-ovarian tissues. Subsequently, the status of caspase expression and function in orchestrating apoptotic cell death in ovarian germ and follicle somatic cells is considered. The most compelling results implicating specific caspases in ovarian function have been derived from mouse single and double knockout model systems. The final outcome of continued studies, in addition to providing information regarding understanding and management of infertility, will influence the development of strategies to treat ovarian cancers and ameliorate the adverse effects of their therapy (for example, chemotherapy).