bluuebunny/crossref_metadata_2025 huggingface dataset:
Morphology and chemical analysis of the sheep conceptus from the 13th to the 19th day of pregnancy
Authors: Wales RG , Cuneo CL
Published: October 2005

Between the 13th and 19th day of pregnancy the sheep conceptus developed into a structure showing considerable differentiation and all the extraembryonic membranes were established. Both length and dried weight of the embryo increased exponentially during this period. A highly significant regression of dried weight on length of embryos was found but measurement of the additional variable, width, did not improve the accuracy of estimating weight from the embryo's dimensions. The mass of the extraembryonic membranes also increased greatly. The dried weight of the trophoblast increased 90-fold over this period; that of the yolk sac increased 17-fold from day 15 to day 19. The protein content of each of the structures making up the sheep conceptus approached 50% of dried weight, which is similar to the proportion in adult soft tissues. The contribution of glycogen to dried weight was low in the sheep embryo and embryonic membranes when compared with estimates in the mouse blastocyst. However, at about the time of implantation the level of this polymer in the embryo was high compared with that in adult soft tissues and approached the level found in adult muscle. Concentrations of DNA and RNA in the sheep conceptus are much higher than the levels in most adult soft tissues and probably reflect higher synthetic rates and a smaller cytoplasmic volume in the embryonic cells.
Luteinizing hormone in the Kowari, Dasyuroides byrnei (Marsupialia:Dasyuridae), during the oestrous cycle and pregnancy, and the effects of gonadectomy in male and female
Authors: Fletcher TP
Published: October 2005

A heterologous double antibody radioimmunoassay has been validated for measurement of LH in plasma of a dasyurid marsupial. Basal concentrations in the oestrous cycle of female kowaris were in the range of 0.5-2.0 ng NIH-oLH-S19 mL-1. Many animals showed elevated LH concentrations (3.0-12.0 ng mL-1) between 8 and 15 days before oestrous but no pre-ovulatory surge was detected. Gonadectomy resulted in greatly increased concentrations of LH, and decreases in testosterone and progesterone concentrations in male and female respectively. In the female LH values ranged from 10-50 ng mL-1 but in the male LH values were greater than 50 ng mL-1.
Incorporation of glucose by the sheep conceptus between days 13 and 19 of pregnancy
Authors: Wales RG , Cuneo CL , Waugh EE
Published: October 2005

Incorporation of glucose into the internal biochemical pools of the sheep embryo and samples of extraembryonic membranes was measured during a 2.5 h incubation in the presence of radiolabelled glucose. Very little glucose was incorporated into the glycogen pools by either the embryo or its membranes and never represented more than 5% of total incorporation. Approximately 65% of label was isolated in the non-glycogen acid-soluble fraction of samples and the remainder was incorporated into non-glycogen macromolecules. The embryonic disc of the day-13 conceptus had the highest rate of incorporation per mg dried weight of any structure studied. Synthesis of non-glycogen macromolecules by the day-13 disc was five to six times that of either day-15 or day-17 embryos. On day 19 very low rates of incorporation into the isolated embryo were found during culture. Evidence suggests that this was a result of limitations on the diffusion of substrate into the embryo because incubation of fragmented embryos produced rates similar to those found on days 15 and 17. Incorporation of glucose into the intracellular pools of extraembryonic membranes per mg dried weight remained relatively low and stable over the period studied and there were only minor differences in the rate of incorporation between membranes. Incorporation of glucose by embryos and extraembryonic membranes was equally as good in phosphate-buffered media as in bicarbonate-buffered solutions and was unaffected by changes in the concentration of lactate and pyruvate in the culture medium.
Production of transgenic merino sheep by microinjection of ovine metallothionein-ovine growth hormone fusion genes
Authors: Murray JD , Nancarrow CD , Marshall JT , Hazelton IG , Ward KA
Published: October 2005

Seven transgenic Merino sheep have been produced by the technique of pronuclear microinjection. Two different Sheep Metallothionein-1a-Sheep Growth Hormone fusion genes were used. Four of the transgenic sheep, all of which contained the gene MTsGH5, did not express the transgene. The remaining three sheep carrying the second fusion gene, MTsGH9, expressed the gene at high levels in a variety of tissues and had elevated blood levels of sheep growth hormone.
In vitro binding of progesterone, cronolone and medroxyprogesterone acetate to uterine progesterone receptors of sheep, rabbit and mouse
Authors: Zhang X , Stone GM , Miller BG
Published: October 2005

Various aspects of the binding of the synthetic progesteongens, cronolone (9 alpha-fluoro-11 beta-hydroxy-17 alpha-acetoxypregn-4-ene-3,20-dione) and medroxyprogesterone acetate (6 alpha-methyl-17 alpha-acetoxypregn-4-ene-3,20-dione, MAP) to uterine cytosol progesterone receptors of the sheep, rabbit and mouse were studied, in an attempt to explain interesting species differences in the biological activity of these steroids. For the sheep, data for binding-site concentration, relative binding affinity (RBA), dissociation constant (Kd) and rates of association and dissociation indicate specific binding of cronolone to the progesterone receptor and these would seem to explain in part the high progestational activity of cronolone in this species. By contrast, with the mouse, there was only a low level of specific binding of cronolone and this appears to explain its inability to maintain pregnancy in this species. Results for the binding activity of cronolone in rabbit uterus were similar to those for the sheep and thus inability of cronolone to maintain pregnancy in the rabbit is not explained by a failure to bind the progesterone receptor. Species differences in binding to the progesterone receptor were also seen with MAP where the RBA, with respect to progesterone, was high in the sheep and rabbit and lower in the mouse. The results, however, do not relate directly to the progestational activity of MAP in these species. Overall, the data indicate that species differences in the binding activity of steroid receptors constitute one factor that causes species-dependent variation in biological responses to progestogens.
Alteration of extracellular cation concentrations and ratios in culture medium does not affect first cleavage division of hamster zygotes in vitro nor overcome the 'two-cell block'
Authors: Bavister BD , Golden M
Published: October 2005

In vivo fertilized hamster one-cell eggs (embryos) were cultured in a simple medium that was modified to provide a wide range of concentrations and ratios of the four major cation components (sodium, potassium, calcium and magnesium) while maintaining total osmotic pressure at 290 +/- 5 mosm. Embryos were cultured in these media to find the optimum cation concentrations for supporting the first cleavage division in vitro and to determine if physiologically abnormal cation concentrations and/or ratios in standard culture media could account for the 'two-cell block' to development in vitro in this species. Despite using a broad range of ratios for sodium:potassium (from 45:1 to 5:1) and for calcium:magnesium (from 17:1 to 1:1), there were no significant differences in the proportions of fertilized eggs that underwent the first cleavage division (approx. 60-80% across all treatments), and none of the two-cell embryos underwent further cleavage during extended culture. These data demonstrate that the first cleavage division of hamster embryos in vitro is insensitive to extracellular concentrations and ratios of the major cations, and that the non-physiological concentrations and/or ratios of these cations in the culture medium are not the primary reason for the failure of hamster zygotes to develop past the two-cell stage in vitro.
Influence of the immature testis on sexual differentiation in the tammar wallaby, Macropus eugenii (Macropodidae: Marsupialia)
Authors: Tyndale-Biscoe CH , Hinds LA
Published: October 2005

Reproduction in the tammar wallaby, Macropus eugenii (Desmarest), is highly seasonal in the females but not the males. This study was designed to determine whether the difference is established during early life as a result of exposure to the developing testes. At day 10 after birth, when the sex can be distinguished externally, testes were removed from males and placed under the flank skin of females, while other groups of males and females were subjected to surgery without interfering with the gonads. The testis grafts remained palpable for 3-6 months. Sex-chromosome constitution was confirmed by karyotyping. At 3 years of age, the body weights and dimensions of the grafted females were not significantly different from those of the sham-operated females, whereas those of the castrated males were significantly larger and were equal to those of the sham-operated males, indicating that there is genetical control of growth independent of the testis in this species of marsupial. During 5 years of observations, none of the grafted females ever produced young, whereas all of the sham-operated females produced young each year from the second year. The grafted females had a mixture of male and female reproductive structures. The pouch and mammary glands developed normally, as did the Mullerian duct derivatives, the vaginal complex, the uteri and the oviducts. The ovaries were either devoid of oocytes and follicles or had reduced numbers, the Wolffian ducts were retained to varying degrees, the urogenital strand had developed into a prostate indistinguishable in size and structure from that of intact males, and the genital tubercle had developed into a normal-sized penis with a crus penis and Cowper's glands. In the castrated males, the scrotum developed normally and contained the gubernaculum and vas deferens. There was no evidence of Mullerian duct derivatives, and the urogenital strand was a simple canal, as in females. There were no Cowper's glands and no penis or erectile tissue. In one hemicastrated male, there was no development of the penis, although the remaining testis occupied the scrotum and showed compensatory hypertrophy. These findings indicate that the testis, at day 10, has a profound influence on the early differentiation of the Wolffian ducts, prostate and penis but cannot influence the differentiation of the Mullerian duct derivatives. The testis does not have any effect on the development of the pouch, mammary glands or scrotum or on somatic growth, all of which are apparently under independent genetical control.
Cortisol feedback on ovine fetal ACTH secretion during the last fifth of gestation
Authors: MacIsaac RJ , McDougall JG , Wintour EM
Published: October 2005

This study investigates the development of the feedback relationship between cortisol and ACTH in ovine fetuses at 117-119 (n = 5), 130-134 (n = 4) and 140-143 (n = 6) days of gestation. During the last 2 h of a 24 h cortisol (100 micrograms h-1) or saline (0.19 mL h-1) infusion, the fetal ACTH response to bolus oCRH injection (10 micrograms) was tested after the collection of basal ACTH and cortisol samples. Basal ACTH concentrations in un-infused or saline-infused fetuses were observed to progressively increase after 117-119 days of gestation. In contrast, a prepartum increase in cortisol concentrations could not be detected until after 130-133 days. Cortisol infusion significantly inhibited basal ACTH values at 130-133 and 140-143 but not at 117-119 days. In the two youngest age groups, cortisol infusion significantly inhibited the fetal ACTH response to oCRH. At 140-143 days, ACTH values after oCRH injection, were elevated to a similar extent during the saline or cortisol infusion. However, at 140-143 days there was evidence to suggest that the ACTH response to oCRH administered during the saline infusion was blunted when compared with earlier stages of gestation. This study suggests that the circulating, basal concentrations of both ACTH and cortisol increase during the last fifth of gestation in the ovine fetus. The exact nature of the prepartum feedback relationship that develops between these two hormones remains to be fully elucidated.
In vitro fertilization, a practical option after failed artificial insemination with donor semen
Authors: Kovacs GT , King C , Rogers P , Wood C , Baker HW , Yates C
Published: October 2005

The progress of 80 couples who failed to conceive by donor insemination was followed through in vitro fertilization cycles using donor sperm. The outcome was better than that for couples undergoing IVF for tubal disease and significantly better than their chance with further cycles of donor insemination.
Production and regulation of progesterone in bovine corpus luteum and placenta in mid and late gestation: a personal review
Authors: Shemesh M
Published: October 2005

In late pregnancy the secretory activity of the corpus luteum of the cow is markedly diminished. This reduced secretion is due to a decline in the number of viable luteal cells as well as reduction in the secretory activity and responsiveness of the cells to trophic agents. The principal extra-ovarian source of progesterone in late gestation appears to be the placenta, especially the fetal cotyledon, which was shown to produce progesterone throughout gestation. Uniquely, this progesterone biosynthesis is cyclic-nucleotide independent, but Ca2+ dependent. It therefore appears that the Ca2(+)-second messenger and protein kinase C systems are responsible for regulation of sterol biosynthesis in the cow placenta.
Effects of conceptus and conceptus secretory products on uterine development in the pig
Authors: Harney JP , Bazer FW
Published: October 2005

Pregnant gilts (n = 6) were subjected to surgery on Day 3 of pregnancy and uterine horns, within gilts, were assigned to either (1) ligation 15 cm below the uterotubal junction (REST) or (2) ligation proximal to the uterine body (NONREST). On Day 15 of pregnancy gilts were hysterectomized and length and weight of both uterine horns were recorded. NONREST uterine horns were longer (P less than .05) than those of REST, but weights were similar. Conceptuses from the REST uterine horns were retarded in development. Morphometric analysis of surface (SE) and glandular (GE) epithelial cells detected no effect of treatment on height, width, area, nuclear area or percentage of nucleus. Porcine conceptus secretory proteins (pCSP) were obtained from medium in which pig conceptuses, collected on Day 15 of pregnancy, were cultured for 30 h. Culture medium was pooled, dialysed and concentrated by Amicon ultrafiltration for intrauterine infusion. Serum proteins (SP) were obtained from blood collected from a Day 15 pregnant gilt and diluted for intrauterine infusion. Catheters were placed into both uterine horns or cyclic gilts (n = 8) on Day 10 of the oestrous cycle and uterine horns were ligated proximal to the uterine body. Gilts received either oestradiol valerate (E2) (5 mg) or corn oil (CO) on Days 11 and 12. On Days 12-14 each gilt received twice daily infusions of Day 15 pCSP in one uterine horn and SP in the other uterine horn.(ABSTRACT TRUNCATED AT 250 WORDS)
Feeding lupin grain for six days prior to a cloprostenol-induced luteolysis can increase ovulation rate in sheep irrespective of when in the oestrous cycle supplementation commences
Authors: Nottle MB , Seamark RF , Setchell BP
Published: October 2005

Three groups of 40 parous Merino ewes were supplemented with 500 g of lupin grain/ewe/day for 7 days starting on either Day 3, 7 or 11 of the oestrous cycle and induced to ovulate by injecting cloprostenol on the sixth day of feeding. Supplementation with lupin grain significantly increased ovulation rate in all groups compared with corresponding controls by increasing the proportion of ewes with twin ovulations. Increases in ovulation rate did not depend on the stage of the cycle at which supplementation began or when during the luteal phase luteolysis was induced. It is concluded that the ovulatory response to lupin grain is initiated near the time of luteolysis.
Intragonadal regulation of immune system functions
Authors: Hedger MP , Qin JX , Robertson DM , de Kretser DM
Published: October 2005

Immune responses within the mammalian gonads, and in particular the testis, are deficient in spite of adequate lymphatic drainage and the presence of lymphocytes and MHC II+ macrophages. There is considerable evidence from in vivo and in vitro studies that this 'suppression' of the immune system may be due, at least in part, to localized inhibition or regulation of normal lymphocyte and/or macrophage functions within the gonads. In the testis, both steroidal and non-steroidal products of the Leydig cells, including androgens, endorphins, and inhibin-related proteins, have been implicated in mediating this activity. In turn, a number of immune cell cytokines affect steroidogenic cell function in vitro. The studies described in this paper indicated that [3H]-thymidine incorporation by adult rat thymocytes in vitro was inhibited by conditioned medium collected from short-term incubations of Percoll-purified adult rat Leydig cells, but stimulated by testicular interstitial fluid and by conditioned medium collected from short-term incubations of adult rat seminiferous tubules. The factors responsible for these effects on thymocyte function appeared to be of large molecular weight, as they were retained by ultrafiltration membranes with exclusion limits of 10,000 or 30,000 daltons. It is hypothesized that an 'immunosuppressive' mechanism, principally mediated by non-steroidal factors secreted by the steroidogenic cells of the gonadal interstitial tissue, exists within the gonads in order to prevent activation of the immune system by germ cell antigens and growth factors associated with germ cell proliferation and differentiation. This mechanism probably acts in parallel with normal antigen-specific tolerance mechanisms operating at the gonadal level. As immune responses to germ cells are believed to be a significant causative factor in infertility, particularly in men, this represents an important area for further study.
A comparison of the effects of bilateral efferent duct ligation and of partial epididymectomy on the testes of rats
Authors: Soler C , Blazquez C , Pertusa J , Nunez M , Nunez J , Nunez A
Published: October 2005

Testis weight as a percentage of body weight did not change following bilateral ligation of the efferent ducts (EDL) close to the epididymis, whereas following removal of part of the epididymis between the site of ligation and a point close to the junction between the caput and corpus (PCE), testis weight first rose linearly until Day 4 and then showed an exponential decrease between Days 4 and 28. After EDL, the perimeter of the seminiferous tubules rose for the first 7 days and then remained elevated, whereas after PCE, there was a linear decrease between Days 4 and 28. Following EDL, the percentage of altered and degenerated tubular cross-sections rose to about 30% and 10%, respectively, during the first 7 days after operation and then remained constant; after PCE, the percentage of altered tubules reached a maximum of 54% by 4 days and then fell, whereas the percentage of degenerated tubules continued to rise to 95% by 28 days. It would appear that all the effects of removal of a portion of the epididymis cannot be explained by blockage of the excurrent ducts, and a specific endocrine effect of the epididymis on the testis is proposed.
Granulocyte macrophage colony stimulating factor (GM-CSF) in the murine reproductive tract: stimulation by seminal factors
Authors: Robertson SA , Seamark RF
Published: October 2005

The activity of GM-CSF during early pregnancy in the murine uterine lumen in vivo and in media conditioned by uterine cells in vitro has been assessed. GM-CSF was detected in uterine luminal fluid recovered by lavage on the morning after syngeneic mating (median level 5.7 CFUc U/uterus) and following mating with vasectomized (5.1 U/uterus) or allogeneic males (4.4 U/uterus), with significantly lesser (P less than 0.05) amounts recovered from the uteri of superovulated, mated mice. By contrast, GM-CSF was only detectable (greater than 0.5 U/uterus) in the luminal fluid of three of 22 unmated oestrous mice examined. No activity was detected in secretions from male accessory glands including seminal vesicle, epididymis, prostate and coagulating gland (less than 0.5 U/gland). GM-CSF was found at higher levels in supernatants from cell monolayers prepared by tryptic digest of the uteri of Day 1 mated mice than those from unmated oestrous mice (P less than 0.05). Little GM-CSF was detected in supernatants from ovariectomized mice. An alpha-GM-CSF polyvalent antibody neutralized the FD5/12 bioassay response confirming the identity of the lymphokine. The interleukins IL-2 and IL-3 were not detected in uterine luminal fluid nor in media conditioned by cell monolayers. We postulate that elevated uterine GM-CSF activity after mating is elicited by a non-sperm associated, non-MHC component of the ejaculate and synthesized by a hormone-responsive endometrial cell population. This cytokine may have an embryotrophic role or contribute to priming of the uterus for implantation.
Immunoglobulin G levels in fetal and newborn tammar wallabies (Macropus eugenii)
Authors: Deane EM , Cooper DW , Renfree MB
Published: October 2005

Immunoglobulin G (IgG) was measured in fetal, neonatal and colostral samples from the tammar wallaby (Macropus eugenii) in order to study the possibility of passively acquired immunity. Samples were obtained from young at a known stage of gestation and at known times (to the minute) after birth. IgG was present (in increasing levels of concentration) in fetal serum, neonatal serum and colostrum. Since the fetus and neonate are probably unable to make immunoglobulin (Ig), it is hypothesized that transplacental and trans-gut transmission takes place from mother to offspring. The vascular yolk sac placenta has a high concentration of IgG, and is the most likely route of transmission from mother to young. Some observations were made of IgA which was found only in colostrum. No Ig of either kind was found in yolk sac fluid.
Effects of a monoclonal antibody against progesterone, on embryo transport, development and implantation in laboratory mice
Authors: Vinijsanun A , Martin L , Wang DY , Fantl VE
Published: October 2005

A monoclonal antibody against progesterone (11P27) given on Day 2 of pregnancy interrupted pregnancy in BALB/c mice but not in BCF1 mice. The reason for this strain difference remains unclear, although it may involve discrimination by the recipient's immune system. The effects in BALB/c mice were reversed by progestin treatment. A dose of 5 nmol, which completely blocked implantation, had no significant effects on embryo transport and development on Day 4. A dose of 10 nmol did not increase the proportion of abnormal embryos, even though it accelerated tubal transport and increased embryo loss. No tubal retention was evident; most remaining embryos reached the uterus at the normal time. The transformation of morulae to blastocysts was only slightly delayed in 11P27-treated mice, and transfer experiments showed no decrease in embryo viability. The antibody appeared to act by blocking actions of endogenous progesterone on the uterus: uteri of 11P27-treated mice failed to develop a decidual cell reaction to intrauterine oil, and embryos from untreated donors failed to implant in 11P27-treated recipients. Antagonism of progesterone by antibody treatment prevented implantation in BALB/c mice apparently by actions on the uterus rather than on the embryo.
Preimplantation development in the streptozotocin-induced diabetic mouse
Authors: Beebe LF , Kaye PL
Published: October 2005

Streptozotocin (STZ) was used to develop a diabetic mouse model in which to study the development of the preimplantation embryo. STZ doses of 0, 160, 190, 210 and 240 mg kg-1 were given; 190 mg kg-1 was found to be the most suitable as the standard diabetogenic dose, providing about 60% mice with plasma glucose greater than 20 mM. The STZ-diabetic mice responded to superovulation with 10 i.u. of gonadotrophin in the same manner as control mice, producing similar embryo numbers at 48 h, 72 h and 96 h post-hCG. Furthermore, the proportion of 2-cell embryos collected from STZ-diabetic mice which developed to blastocysts in vitro was similar to that of 2-cell embryos from control mice. The STZ-diabetic mouse model after superovulation thus produced normal early preimplantation embryos whose development can be examined in detail in a diabetic environment.
Human menstruation and eicosanoids
Authors: Rees MC
Published: October 2005

Menstruation is an event restricted to humans and subhuman primates and in which uterine bleeding and contractions occur. Prostaglandins and leukotrienes are thought to play a major role in the normal process of menstruation as well as in the disorders menorrhagia and dysmenorrhoea.
Second-trimester termination with 16,16 dimethyl-PGE1-methyl ester (gemeprost), compared with a regimen that included intra-amniotic PGF2 alpha and hypertonic saline
Authors: Waldron KW , Renou PM , Lolatgis N , Morris ND , Mamers PM , Oldham J , Healy DL
Published: October 2005

The use of gemeprost (16,16 dimethyl-PGE1-methyl ester) pessaries was compared in an open, randomized single-centre trial with the intra-amniotic injection of PGF2 alpha combined with hypertonic saline, intravenous oxytocin and a hygroscopic cervical dilator (Dilapan) for the termination of pregnancy between 14 and 20 weeks. There was no significant difference in the induction-delivery interval for the two groups. With the exception of an increased incidence of diarrhoea in the gemeprost group, there was no significant difference in other side effects, analgesic requirements or retained placentae. Gemeprost pessaries are an effective alternative to the more invasive methods previously used for the induction of second-trimester termination.
Prostaglandins in dysfunctional labour; evidence for altered production of prostaglandin F2 alpha
Authors: Norman RJ , Reddi K
Published: October 2005

Dysfunctional labour was studied in relation to prostaglandin concentrations in amniotic fluid and production by fetal membranes. Initial clinical validation of the model established the presence of hypokinetic labour with no evidence of obstruction to the fetal progress. Prostaglandin F2 alpha (PGF2 alpha) and 13, 14 dihydro-15-keto-prostaglandin-F2 alpha concentrations in the amniotic fluid were low despite relatively normal concentrations of prostaglandin E2. Membranes removed from patients with the condition released very low concentrations of PGF2 alpha from the amniotic side with no alteration on the choriodecidual side of the membrane. Studies of free and phospholipid-associated arachidonic acid indicated normal release of arachidonic acid in dysfunctional labour. No changes in amniotic fluid-related inhibitors and stimulators of prostaglandin synthetase were detected. It is suggested that PGF2 alpha production is impaired in dysfunctional labour and that this prostaglandin is primarily involved in the progress of labour.
Gestational tissue phospholipase A2 messenger RNA content and the onset of spontaneous labour in the human
Authors: Aitken MA , Rice GE , Brennecke SP
Published: October 2005

Considerable evidence supports a central role for prostaglandins in the genesis of uterine activity and cervical dilatation at the time of human labour. The first step in prostaglandin synthesis is the liberation of arachidonic acid from glycerophospholipids by phospholipase A2. In this study, we examined the periparturitional gene expression of PLA2 in human placentae and fetal membranes, using a cDNA clone for non-pancreatic PLA2. PLA2 gene expression was found to be significantly increased (P less than 0.05) in placentae obtained after spontaneous-onset labour and normal vaginal delivery as compared with placentae from elective Caesarean section. In both of these groups, PLA2 expression in amnion and chorion was significantly less than that in placenta. The results of this study suggest that placental PLA2 gene expression increases in association with spontaneous-onset of labour in the human. This conclusion is consistent with the finding of increased levels of prostaglandins in gestational tissues, amniotic fluid and blood plasma at the time of labour.
Gestational profile of the stimulatory effects of porcine amniotic and allantoic fluids on prostaglandin G/H synthase activity
Authors: Rice GE , Wong MH , Christensen P , Dantzer V , Skadhauge E
Published: October 2005

The effects of porcine fetal fluids (amniotic and allantoic) on microsomal prostaglandin G/H synthase (PGHS) activity were assessed. Both amniotic and allantoic fluids obtained from late-gestation sows stimulated PGHS activity (as indicated by increased formation of radiolabelled prostaglandin) in a dose-dependent manner. At the maximum dose tested, amniotic and allantoic fluids stimulated prostaglandin (PG) formation by 55.5 +/- 1.5 and 58.5 +/- 4.7%, respectively (n = 3, P less than 0.01). Based upon ED50 values, amniotic fluid was approximately threefold more effective than allantoic fluid in stimulating PG formation. The stimulatory effect of amniotic but not allantoic fluid increased significantly (P less than 0.01) during gestation (Days 47-112). The observed changes in the stimulatory effect of amniotic fluid on microsomal PG formation parallels the in vivo changes that occur in intra-uterine PG synthesis. Amniotic fluid stimulatory activity may contribute to this gestational increase in PG synthesis.
In vitro embryo culture in the production of identical merino lambs by nuclear transplantation
Authors: McLaughlin KJ , Davies L , Seamark RF
Published: October 2005

This study examined the viability of embryos developed in vitro from 8- to 16-cell stage blastomeres fused with enucleated oocytes. Of 209 blastomeres recovered and subjected to manipulation and electrofusion procedures, 190 (91%) fused successfully, with 86 (45%) of those undergoing cleavage up to the 4- to 16-cell stage when cultured for 66 h in a synthetic oviduct fluid medium. The viability of the embryos was examined by transferring them to recipient ewes and determining the ewes' pregnancy status by ultrasound on Day 45. Of 86 embryos transferred, 14 developed to fetuses in 8 of the 36 recipients, including four sets of identical twins and one set of quads. In contrast, with uncultured and unmanipulated embryos, 15 fetuses developed from 19 embryos transferred at a similar stage of development. The viability of embryos derived from manipulated zygotes cultured in vitro was comparable to that previously reported for studies employing in vivo culture, indicating the potential of in vitro culture systems based on a simple medium for nuclear-transplantation embryos.
Use of oocytes that failed to be fertilized in vitro to study human sperm-oocyte interactions: comparison of sperm-oolemma and sperm-zona pellucida binding, and relationship with results of IVF
Authors: Liu DY , Lopata A , Baker HW
Published: October 2005

A test for human sperm binding to the oolemma was developed with oocytes that failed to be fertilized in vitro. The zonae pellucidae of the oocytes were removed under a dissecting microscope by brief exposure to dilute HCl (pH 2.5-3.0) in 0.9% NaCl. The zona-free oocytes (ZFOs) were incubated with a mixture of equal numbers of motile sperm from men to be tested and fertile donors. The sperm was differentially labelled with fluorescein or rhodamine and the results expressed as a ratio of the number of test to control sperm bound to several ZFOs in order to control for variability in the ability of the oolemma to bind sperm. The number of sperm bound to the oolemma increased with time and sperm concentration. The sperm-oolemma binding ratio determined for 32 patients undergoing in vitro fertilization (IVF) was significantly correlated with the sperm-zona pellucida (ZP) binding ratio but was not correlated with other sperm tests. The sperm-oolemma binding ratio was also related to the IVF rate, but this was not significant if the sperm-ZP binding ratio was included in the logistic regression model. Only four of the 32 patients had failure of fertilization in vitro. The human sperm-oolemma binding test may be useful for studying the interaction between gametes, but the test is unlikely to be as useful clinically as the sperm-ZP binding test for predicting fertilization in vitro.
Structure and ovarian expression of the oxytocin gene in sheep
Authors: Ivell R , Hunt N , Abend N , Brackman B , Nollmeyer D , Lamsa JC , McCracken JA
Published: October 2005

In sheep, the oxytocin gene is highly up-regulated in the ovarian corpus luteum as well as in the hypothalamus. This expression is already elevated on Day 2 of the oestrous cycle, representing 1% of all transcripts in this tissue, and it declines thereafter to low levels after Day 6 of the cycle. In order to study the mechanisms involved in luteal oxytocin gene expression, we have cloned and sequenced the oxytocin gene from the sheep. This gene is closely homologous to other known mammalian oxytocin genes, especially the bovine one, and comparison of the gene promoter regions highlights several blocks of putative control elements.
Fertility of superovulated ewes following intrauterine or oviducal insemination with fresh or frozen-thawed semen
Authors: Jabbour HN , Evans G
Published: October 2005

Two experiments were conducted with mature Merino ewes to investigate the effects of time and site of insemination of fresh and frozen-thawed semen on the fertility of superovulated ewes. In Experiment 1, each ewe was treated with an intravaginal progestagen sponge and PMSG and/or FSH. They were inseminated in the uterus with fresh or frozen-thawed semen (approximately 100 x 10(6) motile sperm) at 24, 44 or 64 h after sponge withdrawal. Ova were recovered at 88 h after sponge withdrawal and classified as fertilized if they had pronuclei or had cleaved. Mean fertilization rates of recovered ova were 60.0, 93.7 and 87.8% for fresh semen and 46.1, 98.2 and 26.1% for frozen-thawed semen at each of the insemination times (24, 44 and 64 h) respectively. Overall, fertilization rates were higher for fresh semen than for frozen-thawed semen (P less than 0.01), but there was an interaction with time of insemination (P less than 0.01). Following insemination with frozen-thawed semen at 64 h, only 61% of the fertilized ova developed to the 2- to 4-cell stage by the time of embryo recovery; this was less than in any of the other groups (P less than 0.05). In Experiment 2, ewes were inseminated with 100 x 10(6) motile fresh or frozen-thawed semen in the uterus or in the oviducts at 64 h after sponge withdrawal.(ABSTRACT TRUNCATED AT 250 WORDS)
Effect of early ovariectomy and steroid hormone replacement of embryo transport, development and implantation in mice
Authors: Vinijsanun A , Martin L
Published: October 2005

Bilateral ovariectomy on Day 1 of pregnancy increased abnormal embryo numbers on Day 4 and delayed passage of embryos to the uterus. Progestins given on Day 1 reversed these effects; given on Day 3 they reduced numbers of abnormal embryos, but did not restore normal transport. Oestrogen given alone after ovariectomy increased embryo loss, but restored preimplantation embryo development to normal when given on Day 3 after progestins on Day 1. The results suggested that both oestrogen and progesterone were necessary for normal preimplantation embryo development in vivo. However, although Day-1 progestins produced the greatest improvement in embryo transport and preimplantation development, they supported only low implantation rates compared with progestins starting on Day 3, and no progestin treatment returned implantation rates to normal. Sham ovariectomy on Day 1 also reduced implantation rate, suggesting that surgical stress of Day-1 ovariectomy had major adverse effects on embryo viability. This view was supported by experiments involving unilateral ovariectomy, which produced abnormalities in embryo transport, development and implantation, but only on the operated side. Furthermore, the major abnormality induced in embryo development by unilateral and bilateral ovariectomy, viz embryonic autolysis, was not increased in experiments in which pregnancy was blocked by non-surgical antagonism of progesterone. It is concluded that abnormalities in embryo development induced by early ovariectomy are not caused by a deficit of endogenous hormones, but result largely from effects of surgical trauma on oviduct function which can be reversed by treatment with exogenous hormones.
Autoradiographical localization of oxytocin binding sites on ovine oviduct and uterus throughout the oestrous cycle
Authors: Wallace JM , Helliwell R , Morgan PJ
Published: October 2005

A highly specific oxytocin receptor ligand, 125I-labelled d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH9(2)] vasotocin (125I-OTA), was used to localize high affinity oxytocin receptors in ovine uterine and oviduct tissues throughout the oestrous cycle. The pattern of binding revealed by in vitro autoradiography correlated well with the results of the homogenate receptor assays using the same ligand and with previous binding assays using the tritiated ligand. At oestrus, specific 125I-OTA binding was evident on the luminal epithelium of the caruncular and intercaruncular regions, on the epithelial cells lining the secretory uterine glands and in the stroma underlying the caruncular epithelium. In the myometrium diffuse labelling was evident in the outer longitudinal smooth muscle layer. At Day 4 of the cycle, binding to the stroma was diffuse and virtually absent from the glandular epithelium. No specific binding was evident in either tissue at Day 12 of the luteal phase, but by Day 14, prior to the decrease in peripheral progesterone concentrations, binding was again apparent on the luminal epithelium only. Specific binding to the oviduct was localized to the smooth muscle layer of the isthmus region of oestrous ewes and was not detected at any other stage of the oestrous cycle. These studies extend our knowledge of the distribution of oxytocin binding sites in uterine and oviduct tissues throughout the oestrous cycle and suggest that oxytocin has an important role in stimulating oviduct and uterine motility at a time crucial to successful egg collection and/or sperm embryo transport.
Neurophysiological control of the secretion of gonadotrophin-releasing hormone and luteinizing hormone in the sheep--a review
Authors: Thiery JC , Martin GB
Published: October 2005

The anterior pituitary gland secretes pulses of luteinizing hormone (LH) in response to pulses of gonadotrophin-releasing hormone (GnRH) released into the hypophysial portal blood by the hypothalamus. The pulsatile nature of the secretions is very important because the frequency of the pulses is directly related to the activity of the GnRH neurons. We can therefore take advantage of this phenomenon to develop mechanistic interpretations of responses to experimental treatments designed to unravel the neural pathways that influence what is, arguably, the most important individual signal controlling the activity of the reproductive system. We might also resolve the disagreements in the literature covering the neuropharmacology of gonadotrophin secretion. In this review, we describe work towards this end in the sheep. Most (95%) of the 2500 GnRH cell bodies in the sheep brain are located in a region covering the anterior hypothalamus, the medial preoptic area, the diagonal band of Broca, and the septum. The axons of up to 50% of these cells terminate in the organum vasculosum of the lamina terminalis. The remainder terminate in the median eminence and form the final common pathway for the many factors that affect gonadotrophin secretion. Among the factors known to affect the frequency of the pulses (or the activity of the GnRH neurons) are nutrition, pheromones, photoperiod and gonadal steroids (negative and positive feedback). Factors that affect GnRH pulse amplitude are more difficult to determine because variations in pituitary responsiveness prevent the use of LH patterns as a 'bioassay'. Techniques developed recently have allowed the direct measurement of GnRH pulse amplitude and revealed inhibitory effects of oestradiol, but we do not know whether this effect is due to a reduction in the amount of GnRH released by each neurone or a reduction in the number of neurones releasing a pulse. It is unlikely that the factors that alter pulse frequency do so by directly affecting the GnRH cells. For example, it is obvious that other cells, with specific receptors for pheromonal or nutritional stimuli, formulate a signal that is transferred to the GnRH cells via interneurones. Similarly, it is likely that a hypothalamic clock intervenes between photoperiodic inputs and GnRH output. Opioidergic neurons have been proposed as a link in this system, but the complexity of their action makes it unlikely that they directly affect the GnRH neurons. The responses to steroids are simple and rapid, but steroid receptors have not been found in GnRH cells, so at least one other set of interneurones is involved.(ABSTRACT TRUNCATED AT 400 WORDS)
In vivo myometrial activity in the rat during the oestrous cycle: studies with a novel technique of video laparoscopy
Authors: Crane LH , Martin L
Published: October 2005

Previous techniques of recording myometrial activity in vivo gave limited information about the nature of contractions, and disrupted normal reproductive events. To overcome these drawbacks we developed a new in vivo method of video laparoscopy (VL). This involves positioning a laparoscope in the abdomen of anaesthetized rats to view the caudal ends of both uterine horns. Myometrial activity is recorded by video camera onto video tape. Myometrial contractions are classified according to the muscle layers involved, the interaction between layers and the direction of propagation. Experiments with intrauterine balloons and electromyography (EMG) in conscious and ketamine/xylazine anaesthetized rats showed that this anaesthetic does not have major effects on myometrial activity. To validate the VL method, recordings were obtained throughout the oestrous cycle and compared with results obtained with EMG in conscious rats. The frequency and pattern of activity were similar with both techniques although more information was obtained from VL. Frequency of contractions was highest in oestrus and dioestrus and lowest in pro-oestrus, when contractions occurred in groups separated by quiescent intervals. At all stages, longitudinal contractions propagating towards the cervix predominated. Circular muscle activity was only seen at oestrus and dioestrus; that at oestrus consisted of weak peristalses, that at dioestrus was more complex. A major advantage of VL is that it does not interfere with the course of the oestrous cycle, pseudopregnancy or early pregnancy.
Effects of growth factors and hormones on basal and FSH-stimulated inhibin production by porcine granulosa cells in vitro
Authors: Michel U , Ludemann S , Jarry H , Wuttke W
Published: October 2005

The effect of several growth factors, protein and steroid hormones on follicle stimulating hormone (FSH)-stimulated and basal inhibin secretion by mature porcine granulosa cells (g-cells) in culture was examined in order to elucidate the putative role of growth factors and hormones in the regulation of inhibin secretion by porcine g-cells in vitro. Cells were incubated with the respective hormones over a timespan of 0-144 h and immunoreactive inhibin was measured with a radioimmunoassay against porcine inhibin. Epidermal growth factor (EGF) and human transforming growth factor type beta (TGF-beta) decreased basal and gonadotrophin-stimulated inhibin and progesterone in a dose-dependent manner. In the absence of insulin, insulin-like growth factor type I (IGF-I) caused a 4-fold enhancement of basal inhibin secretion, but inhibin secretion was elevated only to 20% above control in the presence of 500 nM insulin. Porcine platelet-derived growth factor (PDGF) had no significant effect on basal or FSH-induced inhibin secretion by g-cells. In addition, neither gonadotrophin-releasing hormone (GnRH) nor prolactin (PRL), arginine vasopressin (AVP) and oxytocin affected basal or FSH-stimulated inhibin release by porcine g-cells. Oestradiol caused a slight but significant (P less than 0.01) rise of basal inhibin production (158% of control) in the last 2 days of culture (96-144 h) and the effect of androstenedione on basal (158% of control) and FSH-stimulated (140% of control) inhibin release (P less than 0.01) was also only visible on Days 4-6 of culture. In contrast to androstenedione and oestradiol, progesterone did not show any effect during 6 days of culture in a dose range of 10(-5) to 10(-9) M. Like steroids, prostaglandin E2 (PGE2) had a stimulatory effect on basal inhibin production (250% of control) by porcine g-cells, visible on Days 3-6 of culture, but an inhibitory effect on FSH-stimulated release (less than 40% of control). Over all the experiments with different hormones and growth factors, tested in varying doses and over a time span of 0-144 h, there was a strong correlation between progesterone and inhibin secretion by g-cells (0-48 h = 0.78; 48-96 h = 0.92; 96-144 h = 0.92). These results suggest that EGF, TGF-beta, IGF-I, oestradiol and androstendione as well as PGE2 have para- and/or autocrine modulatory effects on basal and FSH-stimulated inhibin secretion by mature porcine g-cells in vitro and further demonstrate that the secretion of the proteohormone inhibin and the steroid progesterone are closely related.
A review of the use of progestogen-only minipills for contraception during lactation
Authors: Fraser IS
Published: October 2005

Progestogen-only minipills and other systems for releasing low doses of progestogens alone are widely used for contraception in breast-feeding women around the world. There is good evidence to confirm their acceptability and their lack of effect on milk production, neonatal growth and early development. In contrast, combined oral contraceptives frequently decrease milk production, and may produce minor changes in milk composition. However, even combined oral contraceptives do not appear to produce adverse effects on neonatal well-being and development, although minor reductions in initial growth rate may sometimes occur. Progestogen-only methods may also produce subtle changes in milk composition, although less than combined oral contraceptives. Steroids are transferred from plasma into milk in small quantities, but the amounts are usually very low or insufficient to allow detection in the infants using present-day assays. There has been theoretical concern that these tiny amounts of steroids might affect neonatal reproductive development, but this appears to be unwarranted. Progestogen-only methods are being widely used for post-partum contraception, and they appear to have particular advantages in this situation. They also have few disadvantages; a theoretical concern about a possible effect on later reproductive or sexual development has no evidence to support it. The present licensing situation in Australia, which lists lactation as a relative contraindication to progestogen-only contraceptive use, causes real concern to potential users and appears to lead to frequent errors in compliance.