bluuebunny/crossref_metadata_2025 huggingface dataset:
Pituitary expression of LHβ- and FSHβ-subunit mRNA, cellular distribution of LHβ-subunit mRNA and LH and FSH synthesis during and after treatment with a gonadotrophin-releasing hormone agonist in heifers
Authors: Aspden W. J. , Jackson A. , Trigg T. E. , D'Occhio M. J.
Published: August 2003

The aim was to examine transcriptional and post-transcriptional regulation of LH and FSH biosynthesis. Female cattle were allocated to three groups: (i) Group 1, control (n = 6), synchronized to be at around Day 11 of the oestrous cycle on Day 31; (ii) Group 2 (n = 6), treated with gonadotrophin-releasing hormone (GnRH) agonist (deslorelin) for 31 days; and (iii) Group 3 (n = 6), treated with deslorelin for 28 days. All animals were slaughtered on Day 31. For animals in Group 2, pituitary content of LHβ-subunit mRNA was suppressed 60% (P < 0.001) and LH 95% (P < 0.001), whereas FSHβ-subunit mRNA was suppressed 25% (P > 0.05) and FSH 90% (P < 0.001). Three days after treatment with deslorelin (Group 3) LHβ-subunit mRNA and LH remained suppressed (50% and 95%, respectively; P < 0.001). At the same time, FSHβ-subunit mRNA did not differ from controls (P > 0.05) whereas FSH remained reduced by 80% (P < 0.001). The ratio of LHβ-subunit mRNA present in the nucleus versus cytoplasm of gonadotroph cells was reduced (P < 0.05) in heifers during treatment with deslorelin (0.59 ± 0.05) compared with the ratio in control heifers (1.31 ± 0.22) and heifers 3 days after discontinuation of treatment (1.01 ± 0.05). The findings indicated that treatment with GnRH agonist can suppress LHβ-subunit mRNA expression without any significant effect on FSHβ-subunit mRNA. As LH and FSH contents were suppressed to a greater degree than their β-subunit mRNAs, it would appear that treatment with a GnRH agonist might influence gonadotrophin biosynthesis by a post-transcriptional mechanism(s). For LHβ-subunit mRNA, this would appear not to be reduced export of message from the nucleus.
Effect of dietary intake on steroid feedback on release of luteinizing hormone in ovariectomized cows
Authors: Rhodes F. M. , Chagas L. M. , Clark B. A. , Verkerk G. A.
Published: August 2003

This study tested the hypothesis that the decline in pulsatile release of luteinizing hormone (LH), resulting from steroid negative feedback, is greater in animals fed a low, compared with a high, plane of nutrition. Two-year-old cows were ovariectomized and six days later were fed diets to provide 1.5 × maintenance requirements (n = 6, supplemented) or 0.5 × maintenance requirements (n = 6, restricted) (Round 1). Pulsatile release of LH was measured over a 14-h period on the fifth day of feeding these diets (Day 1); at 6 h, all animals were treated with an intravaginal insert containing 1.38 g progesterone, which remained in place until the end of Day 3. Pulsatile release of LH was again measured for 14 h on Day 3; at 6 h, all animals were injected intramuscularly with oestradiol benzoate (ODB; 1 mg per 500 kg live weight). Three days later, this protocol was repeated, in a cross-over design, with cows that were previously restricted now being supplemented and those cows previously supplemented, now restricted (Round 2). Plasma concentrations of progesterone after intravaginal progesterone treatment were 1.01 ng mL−1 higher in restricted cows compared with supplemented cows (P < 0.001) and were also higher in Round 1 than in Round 2 and on Day 1 than on Day 3 (P < 0.001). Plasma concentrations of oestradiol following injection with ODB did not differ between supplemented and restricted cows (P > 0.1). Dietary intake did not affect mean concentrations of LH, pulse frequency or amplitude during the 6-h period before steroid treatment or the change in these variables following steroid treatment; however, the slope of the decline in concentrations of LH following progesterone treatment was significantly more negative in cows fed restricted diets compared with those fed supplemented diets. In Round 2, mean concentrations of LH were higher preceding, and decreased more following, progesterone treatment compared with the decrease after ODB treatment. In conclusion, acute dietary restriction resulted in a more rapid decline in the release of LH following treatment with intravaginal progesterone, and was associated with higher concentrations of progesterone in plasma.
Determinants of the annual pattern of reproduction in mature male Merino and Suffolk sheep: responses to a nutritional stimulus in the breeding and non-breeding seasons
Authors: Hötzel Maria J. , Walkden-Brown Stephen W. , Fisher James S. , Martin Graeme B.
Published: August 2003

This study was designed to test whether an acute improvement in diet would increase gonadotrophin secretion and testicular growth in strongly photoperiod-responsive Suffolk rams and weakly photoperiod-responsive Merino rams in both the breeding (February–March) and the non-breeding (July–August) seasons. Mature rams (n = 5 or 6) of these breeds were fed a maintenance diet (0.9 kg chaff + 100 g lupin grain) or the same diet supplemented with 1.5 kg lupin grain for 42 days in each season. Lupin grain is a rich source of both energy and protein. Testosterone, luteinizing hormone (LH) and follicle stimulating hormone (FSH) were measured in plasma from blood sampled every 20 min for 24 h on Days −1, 12 and 35 relative to the change in feeding. In rams supplemented with lupins, body mass increased in both breeds in both seasons (P < 0.001). Scrotal circumference and LH pulse frequency increased with lupin supplementation in both seasons (P < 0.003) in Merinos, but only during the breeding season (P < 0.003) in Suffolks. Plasma FSH concentrations were affected by diet only during the breeding season, being elevated on Day 12 in lupin-supplemented rams of both breeds (P < 0.05). It was concluded that Merino rams exhibit reproductive responses to improved nutrition irrespective of time of the year, whereas Suffolk rams respond to nutrition only when the hypothalamic reproductive centres are not inhibited by photoperiod. Thus, Suffolks do respond to nutrition, just as Merinos do, but only when photoperiod allows. This difference between breeds appears to be a result of differences in the neuroendocrine pathways that control pulsatile gonadotrophin-releasing hormone secretion.
The role of insulin-like growth factor II and its receptor in mouse preimplantation development
Authors: Pantaleon M. , Jericho H. , Rabnott G. , Kaye P. L.
Published: August 2003

Insulin-like growth factor II (IGF-II) and its receptor, the IGF-II/mannose-6-phosphate (IGF-II/M6P) receptor, are first expressed from the zygotic genome at the two-cell stage of mouse development. However, their role is not clearly defined. Insulin-like growth factor II is believed to mediate growth through the heterologous type 1 IGF and insulin receptors, whereas the IGF-II/M6P receptor is believed to act as a negative regulator of somatic growth by limiting the availability of excess levels of IGF-II. These studies demonstrate that IGF-II does have a role in growth regulation in the early embryo through the IGF-II/M6P receptor. Insulin-like growth factor II stimulated cleavage rate in two-cell embryos in vitro. Moreover, this receptor is required for the glycaemic response of two-cell embryos to IGF-II and for normal progression of early embryos to the blastocyst stage. Improved development of embryos in crowded culture supports the concept of an endogenous embryonic paracrine activity that enhances cell proliferation. These responses indicate that the IGF-II/M6P receptor is functional and likely to participate in such a regulatory circuit. The functional role of IGF-II and its receptor is discussed with reference to regulation of early development.
Superoxide dismutase affects the viability of thawed European mouflon (Ovis g. musimon) semen and the heterologous fertilization using both IVF and intracytoplasmatic sperm injection
Authors: Berlinguer Fiammetta , Ledda Sergio , Rosati Irma , Bogliolo Luisa , Leoni Giovanni , Naitana Salvatore
Published: August 2003

This study evaluated the effects of superoxide dismutase (SOD) on viability and acrosome integrity of European mouflon spermatozoa after cryopreservation and on the fertilization rates of sheep oocytes after IVF or intracytoplasmatic sperm injection (ICSI). Frozen semen was thawed and washed with synthetic oviduct fluid supplemented with 0.6% bovine serum albumin. After centrifugation, the spermatozoa pellet was split into two culture systems: (i) without SOD; and (ii) in the presence of 1500 IU mL−1 SOD. Sperm viability and acrosome integrity were evaluated simultaneously, immediately after thawing and after 3, 6 and 9 h of culture (5% CO2, 39°C, 90% humidity), by incubating sperm with propidium iodide and fluorescein isothiocyanate-labelled Pisum sativum agglutinin. At the same time, sperm were assessed for motility using a standard scoring system (independent operators’ observation of sperm) that graded degree of motility (i.e. 1 = immotile to 10 = maximum motility, as observed at the moment of thawing). For IVF, frozen–thawed semen derived from the two culture systems was placed in culture together with in vitro-matured sheep oocytes. For ICSI, semen derived from the same culture systems as that for IVF was used, and incubated for 1 h under standard conditions. The results showed a marked difference (P < 0.01) between the percentages of live spermatozoa in medium with SOD and those obtained in medium alone, after 3, 6 and 9 h of culture. The percentages of intact acrosome spermatozoa were higher in medium with SOD after 6 h (P = 0.05) of culture. Spermatozoa motility decreased significantly in SOD containing medium at 3 and 6 h of culture compared with motility in control medium. Fertilization rates were significantly lower in medium with SOD than in medium alone, whereas in the ICSI system fertilization rates were significantly higher in the presence of SOD. The results indicate that the addition of SOD to the culture media enhances the viability rates and the acrosome integrity of cryopreserved mouflon spermatozoa.
Prolactin signalling in porcine theca cells: the involvement of protein kinases and phosphatases
Authors: Ciereszko R. , Opalka M. , Kaminska B. , Górska T. , Dusza L.
Published: August 2003

The hypothesis that protein kinase C (PKC) and tyrosine kinases, as well as serine–threonine and tyrosine phosphatases, are involved in prolactin (PRL) signalling in theca cells harvested from porcine follicles was tested. Theca cells were incubated with PRL for 24 h to stimulate progesterone (P4) production. In addition, treatments included inhibitors of PKC and tyrosine kinases, as well as serine–threonine phosphatase inhibitor and tyrosine phosphatase inhibitor. Prolactin significantly stimulated P4 production by theca cells and all inhibitors suppressed the PRL-stimulated P4 production. After incubation with PRL for 2, 5, 10 or 20 min, theca cells were homogenized and cytosolic and membrane fractions were obtained. This was followed by determination of PKC activity in partially purified subcellular fractions by measuring the transfer of 32P from [γ−32P] adenosine triphosphatase (ATP) to histone III-S. In unstimulated porcine theca cells the major proportion of PKC activity was present in the cytosol. Incubation of cells with PRL resulted in a rapid, time-dependent increase in the amount of PKC activity in the membrane fraction. Protein kinase C activity in the membrane fraction was maximal after 10 min of cells’ exposure to PRL. Protein kinase C activation was assessed also by measuring the specific association of 3H-phorbol dibutyrate (3H-PDBu) with theca cells after treatment with PRL. Prolactin significantly increased 3H-PDBu-specific binding in theca cells. In contrast to PKC, total inositol phosphate accumulation was not affected by PRL in the current study. In summary, PRL stimulated P4 production by porcine theca cells derived from large follicles. The results of the study were consistent with the hypothesis that PKC is one of the intracellular mediators of PRL action in porcine theca cells. Protein kinase C activation does not appear to occur through the action of phosphatidylinositol-dependent phospholipase C. Moreover, the involvement of tyrosine kinases, as well as tyrosine and serine–threonine phosphatases, in PRL signalling in the examined cells is suggested.
Effect of glycosaminoglycans on the preimplantation development of embryos derived from in vitro fertilization and somatic cell nuclear transfer
Authors: Jang Goo , Lee Byeong Chun , Kang Sung Keun , Hwang Woo Suk
Published: August 2003

The purpose of this study was to evaluate the effect of glycosaminoglycans (GAGs) added to the culture medium on the developmental competence of bovine embryos derived from in vitro fertilization (IVF) and from somatic cell nuclear transfer (SCNT). In vitro-matured oocytes were either inseminated with 1 × 106 spermatozoa mL−1 or enucleated and reconstructed with bovine adult ear fibroblasts by SCNT. The embryos were then cultured in modified synthetic oviduct fluid (mSOF) containing 8 mg mL−1 bovine serum albumin (BSA) (control mSOF) or control mSOF supplemented with various GAGs (hyaluronic acid, heparin or chondroitin sulfate) in a dose-dependent manner (0.1, 0.5 or 1.0 mg mL−1). Developmental competence was evaluated by monitoring the numbers of 2-cell embryos, 8–16-cell embryos and blastocysts. The mean cell number of flattened blastocysts stained with 5 μ M bisbenzimide on Day 8 was counted. The percentage of blastocyst formation (IVF and SCNT embryos) from cleaved embryos was significantly higher (P < 0.05) in control mSOF supplemented with 0.5 mg mL−1 hyaluronic acid (45% and 47%), heparin (40% and 47%) or chondroitin sulfate (38% and 44%) compared with control mSOF (30–31% and 30–33%). When compared with the efficacy of 0.5 mg mL−1 GAGs, no significant differences were observed in the developmental competence of both IVF and SCNT embryos. Supplementing control mSOF with 0.5 mg mL−1 GAGs had no effect on the cell number of IVF embryos. In contrast, supplementing 0.5 mg mL−1 of hyaluronic acid, heparin or chondroitin sulfate to control mSOF significantly (P < 0.05) increased the numbers of total cells (93–98 v. 88 cells) and trophectoderm (TE) cells (64–66 v. 55 cells), and decreased the inner cell mass (ICM) to TE cell ratio (48.2–49.8 v. 61.3) in SCNT blastocysts compared with embryos in control mSOF. In conclusion, supplementation of culture media with GAGs may improve the development of bovine IVM–IVF and SCNT embryos to the blastocyst stage. The GAGs increased the quality of blastocysts by increasing total cell numbers in the SCNT embryos.
Expression of collagen XVIII mRNA and protein in human umbilical vein and placenta
Authors: Nasu Kaei , Fujisawa Kayo , Nishida Yoshihiro , Kai Satomi , Sugano Terumasa , Miyakawa Isao , Tateishi Yoshiko
Published: August 2003

Endostatin is a potent angiogenic inhibitor that is derived from collagen XVIII by proteolytic cleavage. Localization of collagen XVIII has been reported in the basement membrane of blood vessels. To examine the involvement of collagen XVIII/endostatin during pregnancy, the distribution of collagen XVIII/endostatin protein in human umbilical vein was evaluated by immunohistochemistry. The expression of collagen XVIII/endostatin in cultured human umbilical vein endothelial cells (HUVEC) was also examined by immunocytochemistry and Northern blot analysis. To examine the release of endostatin in vivo and in vitro, concentrations of endostatin in umbilical venous blood and in HUVEC culture medium were determined using an enzyme-linked immunosorbent assay. Collagen XVIII/endostatin protein was localized to endothelial cells and their basement membrane in the umbilical vein. The expression of collagen XVIII mRNA and protein was detected in HUVEC. However, endostatin was not detected in umbilical venous blood or in HUVEC culture medium. The absence of endostatin release and the presence of its parental protein, collagen XVIII, suggest that the cleavage mechanisms of endostatin might be strongly inhibited under the physiological conditions present during pregnancy. It is therefore considered that vasculature in the feto–placental unit is highly angiogenic, even at the time of parturition.
Lipid metabolism in the embryos of diabetic rats during early organogenesis: modulatory effect of prostaglandin E2
Authors: Sinner Debora , Caviglia J. Matías , Jawerbaum Alicia , Igal R. Ariel , Gonzalez Elida
Published: August 2003

The purpose of this work was to evaluate de novo lipid biosynthesis and the lipid profile, and to study the effect of prostaglandin E2 (PGE2; prostaglandin has previously been found to be involved in diabetes embryopathy) on lipid metabolism in embryos from control and streptozotocin-induced diabetic rats during organogenesis. Increased levels of triacylglycerols were found in embryos of diabetic rats compared with controls, whereas no differences were detected in the levels of cholesterol, cholesterylester, phosphatidylcholine and phosphatidylethanolamine. When the de novo synthesis of lipids in the embryo was studied using [14C]acetate as a tracer, a diminished rate of incorporation of [14C]acetate into the evaluated lipid classes was detected in the diabetic embryo compared with controls. Addition of PGE2 did not modify the incorporation of [14C]acetate into any of the lipid species of control embryos, but enhanced the incorporation of [14C]acetate into triacylglycerol, cholesterylesters, phosphatidylcholine and phosphatidylethanolamine of embryos from diabetic rats. The study’s results show alterations in both synthesis and concentrations of lipids in the embryos of diabetic rats. Interestingly, the results demonstrate that the addition of PGE2, a prostaglandin that reverses the embryonic morphological abnormalities induced by diabetes, prevents disturbances in embryo lipid synthesis caused by diabetes.
Effect of ageing on the proliferation and apoptosis of testicular germ cells in the Syrian hamster Mesocricetus auratus
Authors: Morales E. , Pastor L. M. , Horn R. , Zuasti A. , Ferrer C. , Calvo A. , Santamaría L. , Canteras M.
Published: August 2003

The cellular mechanisms implicated in the atrophy of seminiferous epithelium in ageing are currently under debate, although recent reports suggest that apoptosis may be the primary mechanism implicated in aged germ cell loss. Other investigators have suggested that changes in spermatogonial proliferation are also involved. In the present work, the changes in proliferation and apoptosis in the seminiferous epithelium of aged (24 months) Syrian hamsters were examined in concert and compared with those in young (6 months) animals. Proliferation of germ cells was studied by bromodeoxyuridine labelling and apoptosis was assessed by transmission electron microscopy and in situ TUNEL labelling. Aged animals showed a significant decrease in the numbers of total and proliferating spermatogonia plus preleptotene spermatocytes per unit volume and per testis and in the proliferative index (24.8 ± 1.6%) compared with young animals (30.8 ± 1.2%) (P < 0.05). The number of apoptotic spermatogonia plus spermatocytes per unit volume and the apoptotic index were significantly higher in aged animals (1.51 ± 0.23% v. 0.77 ± 0.04%; P < 0.05). Apoptosis was confirmed by morphological characteristics: condensation of the chromatin and nuclear fragmentation. In aged hamsters, tubular degeneration could be classified into several categories, characterized by maturation arrest and an increase of apoptotic cells in tubular cross-sections in comparison with normal tubular cross-sections. Spermatogonial proliferation was also diminished as seen in tubular cross-sections showing hypospermatogenesis, sloughing off of germ cells and maturation arrest. The results obtained in the present study suggest that the decrease in the proliferation of spermatogonia and the increase in apoptosis constitute two consecutive mechanisms correlated with the ageing of the seminiferous epithelium.
Reproduction and plasma concentrations of leptin, insulin and insulin-like growth factor 1 in growth-hormone-transgenic female sheep before and after artificial insemination
Authors: Kadokawa H. , Briegel J. R. , Blackberry M. A. , Blache D. , Martin G. B. , Adams N. R.
Published: August 2003

The transgenic sheep used in this study expressed an additional copy of the gene for ovine growth hormone (GH), so they had continuously high plasma concentrations of GH. They were used to test whether the GH transgene affected plasma concentrations of the metabolic hormones leptin, insulin-like growth factor 1 (IGF-1) and insulin, and whether these effects were associated with changes in conception, pregnancy or parturition following artificial insemination. Compared with control animals, the GH-transgenic sheep had higher bodyweight, lower body condition score and less subcutaneous fat (Pthinsp;< 0.05). These sheep also had lower plasma concentrations of leptin, higher plasma concentrations of insulin, and higher plasma concentrations of IGF-1 (Pthinsp;< 0.001). A similar proportion of GH-transgenic and control ewes came into oestrus, but the conception rate to artificial insemination was lower in GH-transgenic ewes than in the controls. Only four live lambs were recovered from 12 GH-transgenic ewes (33%) compared with 38 lambs from 43 controls (88%). This outcome was not associated with any difference in plasma progesterone profile in the period leading up to artificial insemination (Day 0). The GH-transgenic ewes had lower concentrations of FSH at all times measured (Day −19, Day −2 and Day 19). These results indicate that appropriate regulation of GH secretion from pituitary or peripheral tissues is necessary for normal reproduction and normal levels of metabolic hormones. Chronically high concentrations of GH were associated with increased levels of IGF-1 and insulin, and decreased levels of leptin.
Pituitary content of luteinizing hormone reveals species differences in the reproductive synchrony between males and females in Australian flying-foxes (genus Pteropus)
Authors: O'Brien G. M. , McFarlane J. R. , Kearney P. J.
Published: August 2003

Flying-foxes (genus Pteropus, suborder Megachiroptera) are long-lived tropical mammals. Their seasonal reproduction appears to be regulated by an endogenous, circannual rhythm modified by multiple environmental cues. Luteinizing hormone (LH) content in pituitary extracts was examined to establish the broad time-frame of pituitary stages in the reproductive seasonality of the flying-foxes. A comparison was made between the grey-headed flying-fox P. poliocephalus, which mates and conceives in autumn, and the little red flying-fox P. scapulatus, which mates and conceives in spring. In P. scapulatus, LH was maximum during the spring mating season at 1494 ng mg−1 in males and 896 ng mg−1 in females. In P. poliocephalus males, LH increased to 1082 ng mg−1 in early summer, 4 months before the mating season; LH concentrations in male P. poliocephalus returned to a low of 222 ng mg−1 by the time of the autumn mating, by which time the female P. poliocephalus expressed elevated LH concentrations (624 ng mg−1). Apparently in P. poliocephalus, the peak LH concentrations in females are delayed by 4 months relative to LH concentrations in males. This is associated with 4 months of energetic courtship on the part of male P. poliocephalus, which is not observed in P. scapulatus, the fertility of which is synchronized between the sexes. The heterologous radioimmunoassay developed using monoclonal antibody 518B7 confirmed classic suppression of LH during pregnancy and lactation in flying-foxes and LH elevation in response to gonadectomy. Juveniles generally had pituitary levels similar to non-breeding levels in adults.
Polyspermic penetration in porcine IVM - IVF systems
Authors: Funahashi Hiroaki
Published: August 2003

Although techniques for in vitro production of porcine embryos have proceeded very rapidly during the past decade, polyspermic penetration still remains a persistent obstacle to porcine in vitro fertilization (IVF) systems. Considerable research on in vitro polyspermic penetration in porcine in vitro-matured (IVM) oocytes has been undertaken to try to solve this problem. In the current paper, recent advancements in overcoming the problems of polyspermy in porcine IVF systems are reviewed. Partial induction of the acrosome reaction of boar spermatozoa in IVF media that contain caffeine is likely to be one of the major causes of polyspermy. A reduction in the number of incompletely acrosome-reacted spermatozoa, which can bind tightly to the zona pellucida and mask free sperm receptors of the zona pellucida, could reduce the incidence of polyspermic penetration; however, morphological differences in the reaction of the zona pellucida have been observed between IVM and ovulated oocytes, which suggests that altered zona morphology may be another cause of polyspermic penetration. It has been shown that the developmental ability of polyspermic porcine embryos to the blastocyst stage is similar to that of normal embryos but that developmental competence to term is much lower. To overcome the current problems of polyspermy, it is suggested that future efforts should be focused on controlling boar sperm function and/or sperm–zona binding to achieve the final maturation associated with normal zona modifications of porcine oocytes at fertilization.
Comparison of maturational and developmental parameters of oocytes recovered from prepubertal and adult pigs
Authors: Ikeda Koji , Takahashi Yoshiyuki
Published: August 2003

To clarify the cause(s) of the differences between the developmental competence of prepubertal and adult porcine oocytes, the following were examined: (i) the meiotic progression, p34cdc2 kinase activity, ooplasm diameter and response to activation stimuli of the oocytes; and (ii) the development of parthenotes and nuclear transfer (NT) embryos obtained using oocytes recovered from prepubertal and adult pigs. Oocytes were recovered from 4- to 8-mm follicles of abattoir-derived ovaries. There were no apparent differences in the morphology of the germinal vesicle, nuclear maturation rate, activity of p34cdc2 kinase or response to parthenogenetic stimulation between prepubertal and adult oocytes. Before and after maturation culture, the ooplasm diameters of prepubertal oocytes were smaller than those of adult oocytes. Parthenotes and NT embryos derived from prepubertal gilt oocytes showed a lower rate of development to the blastocyst stage than those derived from adult oocytes. These results suggest that lower developmental competence of prepubertal oocytes may be caused by their inability to complete ooplasmic maturation, and that this is not because of altered oocyte maturation kinetics and/or p34cdc2 kinase activity. Furthermore, the smaller diameter of the ooplasm of prepubertal oocytes indicates that most oocytes that have routinely recovered from prepubertal gilt ovarian follicles are still in the growing phase and have immature ooplasm.
Selective processing of a follicular matrix metalloproteinase-2 isoform by human oviducal fluid
Authors: Kim Jisoo , Kim Jiyoung , Kim Haekwon , Lee Seung-Jae , Yoon Yong Dal , Kwon Hyuck Chan , Kim Sei Kwang
Published: August 2003

The present study demonstrates that a unique isoform of matrix metalloproteinase (MMP)-2 present in human follicular fluid (FF) can be processed selectively by human oviducal fluid (OF). A gelatin zymogram of untreated FF showed distinct 88-, 84- and 62-kDa gelatinases. Treatment of FF with EDTA resulted in the appearance of 110-kDa gelatinase (GA110). Most gelatinases, except for the 88- and 84-kDa gelatinases, were abolished by pretreatment with EDTA or phenanthroline, but not by pretreatment with a serine/threonine protease inhibitor. When EDTA-pretreated FF was mixed with OF, the GA110 of the FF was specifically reduced. The reduction in GA110 was dependent upon the amount of OF protein and the incubation period after mixing. Treatment of FF with aminophenylmercuric acetate reduced GA110 activity, but this reduction was accompanied by a concomitant increase of 62-kDa gelatinase activity. Anti-human MMP-2 antibody strongly reacted with both GA110 and 62-kDa gelatinases of FF, but only GA110 immunoreactivity was abolished when FF was mixed with OF. The results suggest that the GA110 of FF is an MMP-2 isoform that can be processed selectively by OF.
Relationship between donor animal age, follicular fluid steroid content and oocyte developmental competence in the pig
Authors: Grupen Christopher G. , McIlfatrick Stephen M. , Ashman Rodney J. , Boquest Andrew C. , Armstrong David T. , Nottle Mark B.
Published: August 2003

The developmental competence of oocytes recovered from the ovaries of slaughtered prepubertal and adult pigs was evaluated after in vitro maturation, parthenogenetic activation and culture in vitro. In addition, the effect of prepubertal and adult follicular fluid (FF) on the developmental competence of prepubertal and adult oocytes was investigated. When matured in adult FF, the rates of cleavage (92 v. 73%; P < 0.01) and blastocyst formation (57 v. 38%; P < 0.05) were greater for adult oocytes than for prepubertal oocytes. Blastocysts derived from adult oocytes had more trophectoderm cells (43 v. 30; P < 0.05) and total cells (51 v. 36; P < 0.05) than blastocysts derived from prepubertal oocytes. The developmental competence of prepubertal oocytes was not affected by the FF donor age, whereas the developmental competence of adult oocytes was. Blastocysts derived from adult oocytes matured in adult FF had more trophectoderm cells (38 v. 24; P < 0.005), inner cell mass cells (7 v. 3; P < 0.01) and total cells (45 v. 27; P < 0.001) than blastocysts derived from adult oocytes matured in prepubertal FF. Characterization of the steroid content of the FF used to supplement the maturation medium revealed that adult FF contained more progesterone (42 v. 23 ng mL−1; P < 0.005) and androstenedione (70 v. 16 ng mL−1; P < 0.05) than prepubertal FF. In addition, the molar ratios of progesterone to androstenedione, androstenedione to 17β-oestradiol and androstenedione to testosterone differed (P < 0.05) between prepubertal and adult FF. The results support the hypothesis that a greater proportion of adult oocytes than of prepubertal oocytes has completed ‘oocyte capacitation’. The differences in FF steroid content are indicative of the different follicular environments from which the prepubertal and adult oocytes were isolated, and may be attributed to the observed effects on oocyte developmental competence.
No differences in sheep somatic cell nuclear transfer outcomes using serum-starved or actively growing donor granulosa cells
Authors: Peura T. T. , Hartwich K. M. , Hamilton H. M. , Walker S. K.
Published: August 2003

The aim of this study was to compare serum-starved and non-starved donor cells in sheep nuclear transfer with a special emphasis on cloning outcomes. Sheep oocytes, derived either in vivo or in vitro, were fused with cultured serum-starved or actively growing adult granulosa cells. Resulting blastocysts were transferred to recipients fresh or after vitrification, and subsequent pregnancies followed to term. Donor cell treatment did not significantly affect preimplantation development, pregnancy rates, fetal loss or neonate survival rates. Of 22 lambs born, ten survived the immediate perinatal period but all succumbed at various timepoints within the first few weeks of life. The results of the study suggest that the use of serum-starved cells offers no advantages or disadvantages to cloning outcomes. Neither were significant differences in outcomes observed when using either in vivo- or in vitro-derived oocytes or embryos transferred fresh or after vitrification. Yet, these results continue to highlight problems associated with somatic cell cloning as indicated by offspring mortality. It remains unclear whether the high offspring mortality in the current study was related to species, associated with the cell lines used or the result of other causes.
Oocyte competence in repeat-breeder heifers: effects of an optimized ovum pick-up schedule on expression of oestrus, follicular development and fertility
Authors: Båge Renée , Petyim Sudsaijai , Larsson Birgitta , Hallap Triin , Bergqvist Ann-Sofi , Gustafsson Hans , Rodríguez-Martínez Heriberto
Published: August 2003

Repeat-breeder heifers (RBH) have been shown to present reproductive perturbations during spontaneous cyclicity, which affects oestrus and ovulation. Some of these disturbances (e.g. deviating hormone patterns) are also present during and after cycles of twice-weekly ovum pick-up (OPU), performed according to an optimized schedule allowing normal oestrous cyclicity. In the present study, the effects of OPU on oocyte competence in in vitro maturation (IVM) and in vitro fertilization (IVF) have been evaluated, as were the effects on expression of oestrus and fertility in five RBH (≥4 artificial inseminations) and five virgin heifers (VH controls). In total, 269 RBH and 174 VH oocytes were scored for quality prior to IVM and IVF. The number of follicles available for puncture was higher in RBH, but the oocyte recovery rate after OPU was lower in RBH compared with VH controls and the recovered RBH oocytes were of lower quality, as judged by their appearance at retrieval. Confocal laser scanning and transmission electron microscopy of immature oocytes did not reveal any differences between RBH and VH control oocytes with respect to nuclear and mitochondrial status. However, after IVM, the cytoplasmic spatial reorganization of mitochondria and cortical granules was less advanced in RBH, which could contribute to the subfertility that defines the syndrome. Cleavage rates after IVF were similar in RBH and VH controls. Subsequent to the OPU period, in vivo fertility after controlled artificial insemination was comparable with field fertility rates in both RBH and VH.
Parthenogenetic activation of rat oocytes and their development (in vitro)
Authors: Roh S. , Malakooti N. , Morrison J. R. , Trounson A. O. , Du Z. T.
Published: August 2003

The present study was performed to determine suitable methods for parthenogenetic activation and subsequent development of rat oocytes in vitro. In the first series of experiments, the ability of electrical pulses, strontium, ethanol and ionomycin to activate Sprague-Dawley (SD) rat oocytes was examined. The synergistic effect of strontium and cycloheximide or puromycin was also examined in the second series of experiments. In the third series of experiments, the development of F1 hybrid (SD × Dark Agouti) parthenotes activated with different concentrations of strontium (10–0.08 mM) was compared with that of SD parthenotes. The effect of the timing of activation (10 min and 2, 4 and 6 h after cervical dislocation) was also assessed in a fourth series of experiments. The oocytes activated by strontium showed higher pronuclear formation and cleavage rates than those in the other groups (P < 0.05). Higher blastocyst development was obtained from parthenotes activated by strontium and strontium–cycloheximide compared with the strontium–puromycin group (P < 0.01). However, the total cell number of blastocysts from the strontium–cycloheximide activation group was higher than that of other groups (P < 0.05). With strontium (2.5–10 mM) treatment, 40.9% of blastocysts were obtained from F1 hybrid oocytes, whereas 22.9% were obtained from SD (P < 0.01). The oocytes activated 10 min or 2 h following cervical dislocation showed higher blastocyst development than those of the 4 and 6 h groups (P < 0.01). These results suggest that strontium–cycloheximide produces the highest parthenogenetic activation rate in the rat and that oocytes must be activated by 2 h after cervical dislocation.
Fetal mortality diagnosis by ultrasound in the vicuña (Vicugna vicugna)
Authors: Aller Juan F. , Rebuffi G. E. , Cancino A. K. , Alberio R. H.
Published: August 2003

Ultrasonography is widely used in domestic species of camelids, but there is no information about the use of this technique for pregnancy diagnosis and determination of embryonic or fetal losses in the vicuña (Vicugna vicugna). The study was performed in 202 vicuñas (3-year-old females, n = 31; adult females, n = 171) mated during the summer months (January through March 2001) at the Abra Pampa Experimental Farm of Altitude in north-west Argentina. Transrectal ultrasound examination was performed in May (estimated 40–120 days of gestation) to determine the number of pregnant females. The pregnancy rate was 45.5% (92/202). No significant difference (P > 0.05) was observed between the pregnancy rate of 3-year-old females (41.9%) and adult females (46.2%). In December (estimated 250–330 days of gestation) of the same year, a second ultrasonographic study was performed on those vicuñas that were diagnosed as pregnant from the first ultrasound scan. Of 92 animals diagnosed as pregnant in May, only 84 were present in December, because eight females died in the period of study. Overall, 11.9% (10/84) of fetuses were lost during the period (18.1% in 3-year-old vicuñas and 10.9% in adult vicuñas). In conclusion, transrectal ultrasonography was found to provide a rapid and non-invasive means for pregnancy and fetal mortality diagnosis in vicuñas.
Expression of hyaluronan synthase-3 in porcine oviducal epithelium during oestrus
Authors: Tienthai Paisan , Kimura Naoko , Heldin Paraskevi , Sato Eimei , Rodriguez-Martinez Heriberto
Published: August 2003

Hyaluronan (HA) has been related to fertilization and embryo development in the pig. Furthermore, HA is present in pig oviduct fluid and the lining epithelium, particularly of the pre-ovulatory sperm reservoir. Because the mechanisms that regulate HA synthesis have not yet been clarified, semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) was conducted to assess the expression of mRNAs of two HA-synthesizing enzymes (has2 and has3) in the oviduct epithelium (uterotubal junction, isthmus, ampullary–isthmic junction and ampulla segments) of non-inseminated (control) and inseminated (treatment) sows at pre-, peri- and post-ovulatory oestrus. Only has3 mRNA was detected; it was present in all tubal segments of both control and treatment samples. The level of has3 expression did not vary significantly between non-inseminated and inseminated specimens, but there was a tendency (NS) for increased mean values during the peri- and post-ovulatory stages compared with pre-ovulation. It is concluded that has3 is expressed by the porcine endosalpinx epithelium and the levels of expression do not vary during the critical periods of sperm transport and fertilization, despite fluctuating levels of HA in the tubal fluid at corresponding periods.
Seasonal reproduction and delayed sexual maturity in mound-building mice Mus spicilegus
Authors: Gouat Patrick , Féron Christophe , Demouron Simone
Published: August 2003

In the mound-building mouse, Mus spicilegus, which is found from Central to Eastern Europe, reproduction is seasonal and limited to spring and summer. In autumn, the mice build voluminous mounds composed of vegetable matter covered with earth, where juvenile animals (autumnal individuals) over-winter in groups without reproducing. Autumnal animals delay reproduction until the next spring when they are 6 months old. The influence and interactions of environmental (short light period and cold temperature (C conditions) compared with long light period and temperate temperature (T conditions)) and social factors (lack of odours from breeding adults (NB conditions) compared with presence of odours from breeding adults (B conditions)) on reproduction and sexual maturation were studied. Forty groups of three autumnal individuals (two males and a female or two females and a male) were placed in four experimental conditions (CB, CNB, TB and TNB), corresponding to interactions between environmental and social factors (n = 10 groups for each condition). Of the 40 groups only one initiated reproduction during the 18 weeks of cohabitation. Subsequently, animals were separated and isolated for 1 month and then paired with unfamiliar partners. Reproduction was monitored for an additional month, and 24 out of 39 females reproduced. In addition, of eight reproducing pairs placed in C conditions and 10 reproducing pairs maintained in T conditions, all but one pair continued reproduction. It was concluded that the delay in reproduction observed in autumnal individuals was the result of the social effects of living in groups as opposed to the environmental conditions of winter.
Reduced polyspermic fertilization of porcine oocytes utilizing elevated bicarbonate and reduced calcium concentrations in a single-medium system
Authors: Herrick J. R. , Conover-Sparman M. L. , Krisher R. L.
Published: August 2003

The development of efficient systems for in vitro production of porcine embryos has been hampered by a high incidence of polyspermic fertilization. A recently developed single-medium system for porcine in vitro maturation (IVM), IVF and in vitro embryo culture (IVC) (Purdue Porcine Medium; PPM) was modified with elevated bicarbonate (44 mM) and reduced calcium concentrations (1.7 mM) for IVF (PPMfert.2). Oocyte penetration was evaluated after maturation in PPMmat (0.5 mg mL−1 hyaluronan, 0.6 mM cysteine, 10 ng mL−1 epidermal growth factor (EGF), 0.1 U mL−1 porcine LH and FSH, and 1 × Minimal Essential Medium (MEM) vitamins) and fertilization (5 h with 5 × 105 sperm mL−1) in either PPMfert.2 or mTBM (20 mM Tris, 0.0 mm bicarbonate, 7.5 mM calcium). Embryonic development (cleavage and blastocyst stages) was assessed after culture in PPM1 and PPM2. Although penetration was lower in PPMfert.2 (69.9%) compared with mTBM (83.9%), 48.8% of penetrated oocytes were fertilized normally in PPMfert.2 compared with only 27.8% normal fertilization in mTBM. More oocytes cleaved in PPMfert.2 (77.9% v. 53.7%), but development to the blastocyst stage was not different between treatments (14.1% v. 14.3%). Further work is needed to improve embryonic development, but reduced polyspermic penetration is an important step in the optimization of the PPM system for in vitro porcine embryo production.
Ubiquitin-specific protease activity of USP9Y, a male infertility gene on the Y chromosome
Authors: Lee Kyung Ho , Song Gyun Jee , Kang Inn Soo , Kim Soo Woong , Paick Jae-Seung , Chung Chin Ha , Rhee Kunsoo
Published: August 2003

Deletions of USP9Y have been observed among infertile males with defective spermatogenesis. Therefore, the gene has been designated as a male infertility gene on the Y chromosome. However, it remains to be determined how male infertility results from deletions of this gene. In order to initiate an investigation into the cellular functions of USP9Y in male germ cell development, in the present study we characterized the enzymatic specificity of USP9Y. Our results show that both USP9Y and Fam, the mouse infertility protein Usp9x, possess a protease activity specific to ubiquitin. These results suggest that, through de-ubiquitination, USP9Y may stabilize a specific target protein that is important for male germ cell development.
Nuclear morphology, diameter and meiotic competence of buffalo oocytes relative to follicle size
Authors: Yousaf Muhammad Rizwan , Chohan Kazim Raza
Published: August 2003

The nuclear morphology, diameter and in vitro meiotic competence of buffalo oocytes was compared relative to follicle size. Cumulus–oocyte complexes (COCs) were collected from 1–<2, 2–<3, 3–<4, 4–<6 and 6–<8 mm follicles from abattoir ovaries. Cumulus cells were removed using 3 mg mL−1 hyaluronidase in saline and repeated pipetting. Denuded oocytes were measured, fixed in 3% glutaraldehyde, stained with 4,6-diamidoino-2-phenylindole and evaluated for nuclear morphology, namely the stage of germinal vesicle (GV) development before in vitro maturation (IVM). The COCs from >2-mm follicles were matured in vitro in their respective size groups for 24 h in Medium 199 supplemented with 10 μg mL−1 follicle-stimulating hormone, 10 μg mL−1 luteinizing hormone, 1.5 μg mL−1 oestradiol, 75 μg mL−1 streptomycin, 100 IU mL−1 penicillin, 10 mM HEPES and 10% fetal bovine serum. Matured oocytes were fixed, stained and evaluated for GV status and meiotic development. The number of oocytes collected from follicles 1–<8 mm in diameter averaged 1.82 per ovary. Oocytes from follicles 1–<2 mm (107.7 ± 1.6 μm), 2–<3 mm (108 ± 1.1 μm) and 3–<4 mm (114.6 ± 1.3 μm) in diameter were smaller in diameter (P < 0.05) than oocytes from follicles 4–<6 mm (124.4 ± 1.3 μm) and 6–<8 mm (131.9 ± 1.4 μm) in diameter. A majority of oocytes (P < 0.05) from <4-mm follicles was at the initial stages of GV development (GV-I, II and III), whereas oocytes from 4–<6- and 6–<8-mm follicles were at the final stages of GV-IV (35.0 and 21.6% respectively) and GV-V (49.1 and 67.5% respectively). Poor IVM rates of 32.0% and 32.7% to metaphase (M)-II were observed for oocytes isolated from 2–<3- and 3–<4-mm follicles, respectively, whereas significantly (P < 0.05) more oocytes from 4–<6- and 6–<8-mm follicles reached M-II (67.1% and 79.1% respectively). In conclusion, buffalo oocytes displayed a size-dependent ability to undergo meiotic maturation and we suggest that oocytes from >4-mm follicles should be considered in buffalo in vitro fertilization systems for better results.
Insulin-like growth factor binding proteins in follicular fluid from morphologically distinct healthy and atretic bovine antral follicles
Authors: Irving-Rodgers H. F. , Catanzariti K. D. , Master M. , Grant P. A. , Owens P. C. , Rodgers R. J.
Published: August 2003

In bovine follicles 2–5 mm in diameter, two morphologically distinct types of healthy follicles and two types of atretic follicles have been described recently. Healthy follicles either have columnar basal granulosa cells with follicular basal lamina composed of many layers or ‘loops’ or they have rounded basal cells with a conventional single-layered, aligned follicular basal lamina. In atretic follicles, cell death either commences at the basal layer and progresses to the antrum (basal atresia) with macrophage penetration of the membrana granulosa or death progresses from the antrum in a basal direction (antral atresia). Little is known about how these different phenotypes develop. To determine whether insulin-like growth factor binding protein (IGFBP) levels in follicular fluid differ between these different types of follicles, we measured IGFBP levels in fluids from these follicles. A total of 61 follicles were assessed by light microscopy and characterized by morphological analysis as either healthy, with columnar or rounded basal granulosa cells, or as undergoing antral or basal atresia. The IGFBP concentration in the follicular fluid of individual follicles from the four groups (n = 12–20 per group) was identified by Western ligand blots using 125I-insulin-like growth factor (IGF)-II as a probe. Insulin-like growth factor binding proteins 2, 3 (44 and 40 kDa), 4 (glycosylated and non-glycosylated) and 5 were observed. The levels (per volume of fluid) of IGFBPs 2, 4 and 5 were greater in atretic follicles than in healthy follicles. However, there were no statistical differences in levels of each IGFBP between either the two types of healthy follicle or between the two types of atretic follicles. Thus, IGFBP levels are not related to the different types of healthy or atretic follicles.