Serum profiles of luteinizing hormone,progesterone and total estrogens during the canine estrous cycle

Serum levels of LH, total estrogen and progesterone were measured daily by radioimmunoassay during proestrus, estrus and early diestrus in five beagle bitches. Occurrence of the LH peak relative to the onset of estrus was quite variable ranging from 3 days before to 7 days after the onset of estrus. Serum LH levels were elevated for 3 days with a peak value of 25 ± 2 ng/ml reached 2.4 days after the start of estrus. LH levels were ≤ 2 ng/ml when measured at other times during the estrous cycle. Estrogen titers ranged from 84 ± 39 pg/ml at 9 days before the LH peak to 175 ± 15 pg/ml coincident with the LH peak. A broad estrogen peak was evident beginning 5 days before and continuing for 5 days after the LH peak. An estrogen surge was seen in 4 of 5 dogs immediately preceding or coincident with the LH peak suggesting that LH release in the bitch is triggered by a sharp elevation in estrogen levels. Serum progesterone levels rose from ≤ 5 ng/ml before the LH peak to 46 ± 6 ng/ml 6 days afterwards.     

Increased appearance of inducible nitric oxide synthase in the uterus and embryo at implantation.

The aim of this study was to investigate the presence of iNOS in the murine uterus and embryo at implantation. Western blot analysis showed the presence of a 130-kDa band with strong reactivity to anti-iNOS antibody in the pre- and peri-implantation stage uteri. This band was faint in the postimplantation uteri. Immunocytochemical studies showed a heavy localization of iNOS specifically on the apical cells of the uterine endometrium in the pre- and peri-implantation stages. But the postimplantation uteri showed resorbed endometrium showing weaker expression of iNOS. The iNOS was induced by estrogen and the induction was intensified when progesterone was given along with estrogen. This truly mimics the in vivo situation since implantation in mice occurs when an estrogen surge occurs on a background of progesterone. The embryos too express iNOS at the peri-implantation stage. We suggest that iNOS expressed at peri-implantation would lead to enhanced NO production, which could act as a vasodilator and an angiogenic mediator. These effects could promote the attachment of the blastocyst to the uterus.     

Prolactin and delay of implantation in lactating adrenalectomized rats

Adrenalectomy before pregnancy in rats caused the persistence of high blood levels of prolactin (PRL) throughout the ensuing postpartum lactation. The persistence of hyperprolactinaemia was without effect on the (delayed) timing of blastocyst implantation during lactation. The findings indicate that ovarian cycles and pregnancy may continue normally despite the absence of adrenal hormones. They reveal that the enhanced release of pituitary PRL in response to suckling is not dependent on the removal of the adrenals during early lactation. The normal delay of blastocyst implantation through suckling, in the presence of abnormally high concentrations of PRL in blood, throws doubt on the supposed critical role of PRL in determining the length of the period of delay of implantation during lactation.     

Lipid signaling in embryo implantation

A reciprocal interaction between the implantation-competent blastocyst and the receptive uterus is required for successful implantation. Although various molecular pathways are known to participate in this cross-talk, a comprehensive understanding of the implantation process is still missing. Gene expression studies and genetically engineered mouse models have provided evidence that lipid mediators serve as important signaling molecules in coordinating the series of events during early pregnancy including preimplantation embryo formation and development, implantation and postimplantation growth. This review focuses on the roles of two groups of lipid mediators, prostaglandins (PGs) and endocannabinoids, during early pregnancy. Our laboratory has shown that while PGs generated by the cPLA2-cyclooxygenase (COX) system are essential to ovulation, fertilization, and implantation, endocannabinoids are important for synchronizing preimplantation embryo development with uterine receptivity for implantation. A better understanding of these molecular signaling pathways is hoped to generate new strategies to correct implantation failure and improve pregnancy rates in women.     

Photoperiod control of birth timing in the harbour seal (Phoca vitulina)

The harbour seal ( Phoca vitulina ) has delayed implantation, precise annual birth timing, and significant latitudinal variation in birth timing. The birth timing patterns of four distinct groups of seals, including colonies of P. v. vitulina and colonies and captive individuals of P. v. richardsi , were examined using population-based photoperiod analysis to assess the role of photoperiod in setting annual birth timing. This analysis simultaneously determined the time, relative to birth, at which photoperiod response was likely to occur and the critical photoperiod.
Despite marked differences in birth timing patterns, a high level of agreement was found among groups for the timing of photoperiod response. The two subspecies, however, demonstrated significantly divergent critical photoperiods. Eastern Atlantic harbour seals were exposed to a common critical photoperiod of 11.7 h/day on the 268th pre-partum day. Wild Pacific harbour seals were exposed to 14.3 h/day on the 283rd pre-partum day. These times corresponded to the estimated occurrence of blastocyst implantation.
Using the above information, three small captive populations were subjected to artificially prolonged photoperiods during the period of embryonic diapause to test whether subsequent birth timing could be delayed. Technical difficulties invalidated results at two sites. At the third and largest colony, the mean pupping date of six individuals was significantly delayed by 10.7days.
The precision and latitudinal variation of annual birth timing in the harbour seal are due to a response to photoperiod which occurs immediately prior to blastocyst implantation. The critical photoperiod, however, is divergent among subspecies and, thus, has probably evolved allowing seasonal adaptation. Similar environmental signalling has been described for California sea lions and northern fur seals and represents the likely timing mechanism for most pinniped species.     

Dual source and target of heparin-binding EGF-like growth factor during the onset of implantation in the hamster

Heparin binding EGF-like growth factor (HB-EGF), encoded by the Hegfl gene, is considered as an important mediator of embryo-uterine interactions during implantation in mice. However, it is unknown whether HB-EGF is important for implantation in species with different steroid hormonal requirements. In mice and rats, maternal ovarian estrogen and progesterone (P(4)) are essential to implantation. In contrast, blastocyst implantation can occur in hamsters in the presence of P(4) alone. To ascertain whether HB-EGF plays any role in implantation in hamsters, we examined the expression, regulation and signaling of HB-EGF in the hamster embryo and uterus during the periimplantation period. We demonstrate that both the blastocyst and uterus express HB-EGF during implantation. Hegfl is expressed solely in the uterine luminal epithelium surrounding the blastocyst prior to and during the initiation of implantation. Hypophysectomized P(4)-treated pregnant hamsters also showed a similar pattern of implantation-specific Hegfl expression. These results suggest that uterine Hegfl expression at the implantation site is driven by either signals emanating from the blastocyst or maternal P(4), but not by maternal estrogen. However, in ovariectomized hamsters, uterine induction of Hegfl requires the presence of estrogen and activation of its nuclear receptor (ER), but not P(4). This observation suggests an intriguing possibility that an estrogenic or unidentified signal from the blastocyst is the trigger for uterine HB-EGF expression. An auto-induction of Hegfl in the uterus by blastocyst-derived HB-EGF is also a possibility. We further observed that HB-EGF induces autophosphorylation of ErbB1 and ErbB4 in the uterus and blastocyst. Taken together, we propose that HB-EGF production and signaling by the blastocyst and uterus orchestrate the 'two-way' molecular signaling to initiate the process of implantation in hamsters.     

Ginkgolides induce apoptosis and decrease cell numbers in mouse blastocysts

In this report, we examine the cytotoxic effect of ginkgolides, the major components of Ginkgo biloba extracts, on the blastocyst stage of mouse embryos and on subsequent early postimplantation embryonic development in vitro. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay revealed that blastocysts treated with 5 or 10muM ginkgolide A or ginkgolide B showed increased apoptosis versus untreated controls. This could be correlated with the observation that ginkgolide-treated blastocysts showed a significant reduction in the average number of total cells in the blastocyst and trophectoderm/inner cell mass lineage versus controls. In addition, ginkgolide-pretreated blastocysts showed normal levels of implantation on culture dishes in vitro, but significantly fewer embryos reached the later stages of embryonic development in the treatment groups versus the controls, instead dying at relatively early stages of development. Our results collectively indicate that ginkgolide treatment of mouse blastocysts induces apoptosis, decreases cell numbers, retards early postimplantation blastocyst development, and increases early-stage blastocyst death. These novel findings provide important new insights into the effect of Ginkgo biloba extracts on mouse blastocysts.     

Dysregulation of EGF family of growth factors and COX-2 in the uterus during the preattachment and attachment reactions of the blastocyst with the luminal epithelium correlates with implantation failure in LIF-deficient mice

Various mediators, including cytokines, growth factors, homeotic gene products, and prostaglandins (PGs), participate in the implantation process in an autocrine, paracrine, or juxtacrine manner. However, interactions among these factors that result in successful implantation are not clearly understood. Leukemia inhibitory factor (LIF), a pleiotropic cytokine, was shown to be expressed in uterine glands on day 4 morning before implantation and is critical to this process in mice. However, the mechanism by which LIF executes its effects in implantation remains unknown. Moreover, interactions of LIF with other implantation-specific molecules have not yet been defined. Using normal and delayed implantation models, we herein show that LIF is not only expressed in progesterone (P4)-primed uterine glands before implantation in response to nidatory estrogen, it is also induced in stromal cells surrounding the active blastocyst at the time of the attachment reaction. This suggests that LIF has biphasic effects: first in the preparation of the receptive uterus and subsequently in the attachment reaction. The mechanism by which LIF participates in these events was addressed using LIF-deficient mice. We observed that while uterine cell-specific proliferation, steroid hormone responsiveness, and expression patterns of several genes are normal, specific members of the EGF family of growth factors, such as amphiregulin (Ar), heparin-binding EGF-like growth factor (HB-EGF), and epiregulin, are not expressed in LIF(-/-) uteri before and during the anticipated time of implantation, although EGF receptor family members (erbBs) are expressed correctly. Furthermore, cyclooxygenase-2 (COX-2), an inducible rate-limiting enzyme for PG synthesis and essential for implantation, is aberrantly expressed in the uterus surrounding the blastocyst in LIF(-/-) mice. These results suggest that dysregulation of specific EGF-like growth factors and COX-2 in the uterus contributes, at least partially, to implantation failure in LIF(-/-) mice. Since estrogen is essential for uterine receptivity, LIF induction, and blastocyst activation, it is possible that the nidatory estrogen effects in the P4-primed uterus for implantation are mediated via LIF signaling. However, we observed that LIF can only partially resume implantation in P4-primed, delayed implanting mice in the absence of estrogen, suggesting LIF induction is one of many functions that are executed by estrogen for implantation.     

Luteal function during the periimplantation period and requirement for estrogen for implantation and pregnancy maintenance in the non-human primate

An attempt has been made in this paper to review our present understanding of luteal function during the periimplantation period and in particular hormonal requirement for implantation and maintenance of early pregnancy in the non-human primate.In a fertile cycle thecorpus luteum is apparently rescued from luteolysis by chorionic gonadotropin secreted by the implanted blastocyst, In the bonnet monkey the serum progesterone titers during the luteal phase of a fertile cycle seems higher compared to that of nonmated cycling monkeys. This suggested that thecorpus luteum is receiving some stimulatory signal from the blastocyst even prior to implantation. The recent demonstration that human blastocyst in culture secretes into the medium human chorionic gonadotropin essentially support the above assumption. However, attempts to extend the luteal phase of cycling unmated monkeys with exogenous human chorionic gonadotropin injection has hitherto not met with complete success suggesting that there could be other than chorionic gonadotropin, additional luteal stimulatory factors the unimplanted blastocyst is secreting.Corpus luteum is the principle source of both progesterone and estrogen produced during the periimplantation period and dysruption of luteal function, brought about by either lutectomy or ovariectomy or luteinizing hormone antiserum treatment, followed by progesterone supplementation leads to maintenance of pregnancy. This has lead to questioning the need for estrogen in the maintenance of early pregnancy. Recent work using Zuclomiphene, an antiestrogen during days 5–11 of cycle in rhesus monkeys mated between day 9–14, has however, suggested that estrogen may be required for implantation. Further work is needed to arrive at an unequivocal decision regarding the need for estrogen in maintenance of early pregnancy in the primate.     

Polarity of the mouse embryo is anticipated before implantation

In most species, the polarity of an embryo underlies the future body plan and is determined from that of the zygote. However, mammals are thought to be an exception to this; in the mouse, polarity is generally thought to develop significantly later, only after implantation. It has not been possible, however, to relate the polarity of the preimplantation mouse embryo to that of the later conceptus due to the lack of markers that endure long enough to follow lineages through implantation. To test whether early developmental events could provide cues that predict the axes of the postimplantation embryo, we have used the strategy of injecting mRNA encoding an enduring marker to trace the progeny of inner cell mass cells into the postimplantation visceral endoderm. This tissue, although it has an extraembryonic fate, plays a role in axis determination in adjacent embryonic tissue. We found that visceral endoderm cells that originated near the polar body (a marker of the blastocyst axis of symmetry) generally became distal as the egg cylinder formed, while those that originated opposite the polar body tended to become proximal. It follows that, in normal development, bilateral symmetry of the mouse blastocyst anticipates the polarity of the later conceptus. Moreover, our results show that transformation of the blastocyst axis of symmetry into the axes of the postimplantation conceptus involves asymmetric visceral endoderm cell movement. Therefore, even if the definitive axes of the mouse embryo become irreversibly established only after implantation, this polarity can be traced back to events before implantation.     

Alaskan marine mammal tissue archival project

A project to establish an archive of Alaskan marine mammal tissues was conceived in 1987 to be a part of the National Biomonitoring Specimen Bank (NBSB). Protocols and field collection of marine mammals, long-term storage, and analysis are summarized in this paper. Instrumental neutron activation analysis has been used for an initial evaluation of trace element content in samples of northern fur seal (Callorhinus ursinus) from the Pribilof Islands. The findings agree with previously observed trace element levels in northern fur seals. The archived specimens can be used in future studies when comparisons of past and present pollution levels are needed.

     

Fur seals at Macquarie Island: post-sealing colonisation,trends in abundance and hybridisation of three species

Commercial sealers exterminated the original fur seal population at Macquarie Island in the early 1800s. The first breeding record since the sealing era was not reported until March 1955. Three species of fur seal now occur at Macquarie Island, the Antarctic (Arctocephalus gazella), subantarctic (A. tropicalis) and New Zealand (A. forsteri) fur seal. Census data from 54 breeding seasons in the period 1954–2007 were used to estimate population status and growth for each species. Between the 1950s and 1970s, annual increases in pup production for the species aggregate were low. Between 1986 and 2007, pup production of Antarctic fur seals increased by about 8.8% per year and subantarctic fur seals by 6.8% per year. The New Zealand fur seal, although the most numerous fur seal species on Macquarie Island, has yet to establish a breeding population, due to the absence of reproductively mature females. Hybridisation among species is significant, but appears to be declining. The slow establishment and growth of fur seal populations on Macquarie Island appears to have been affected by its distance from major population centres and hence low immigration rates, asynchronous colonisation times of males and females of each species, and extensive hybridisation.     

Postimplantation development of mitomycin C-treated mouse blastocysts

Treatment of morula-stage mouse embryos with mitomycin C (0.004-0.5 microgram/ml) in vitro resulted in a decrease in the number of inner cell mass (ICM) cells at the blastocyst stage. The trophectoderm population was reduced only at the highest dosage (0.5 microgram/ml) tested. Postblastocyst development in vitro was retarded: Fewer embryos formed trophoblastic outgrowth, and the ICM was poorly developed. The embryo transfer experiments demonstrated that a reduction in ICM cell numbers diminished the potential of embryogenesis. The presence of a sufficient number of trophoblasts and ICM cells in the blastocyst is therefore a prerequisite for successful implantation and embryogenesis. The mitomycin-treated blastocysts with only 70% of normal ICM cells developed to egg cylinders that were about half normal size, but by days 12-14 the body size of the surviving embryo was similar to that of the control embryo. Morphogenesis was retarded during the early organogenesis stages, but only a slight delay was seen in the treated embryo on day 12. Such observation strongly suggests that a restorative phase of growth and morphogenesis has occurred during the immediate postimplantation period.     

Morphological changes in the blastocyst of the western spotted skunk during activation from delayed implantation

Blastocysts collected from the spotted skunk during delay of implantation, early activation and late activation demonstrate three-tiered growth and developmental changes. The slow-growing blastocyst from the several months of delay is small (less than 1.1 mm) with a rounded inner cell mass consisting of clusters of rounded, lipid-filled cells. During the several days of early activation, the lipid in both inner cell mass and trophoblast diminishes, polyribosomes increase in number, and the endodermal layer differentiates as the blastocyst grows (1.2-1.6 mm). At activation the inner cell mass flattens, becomes uncovered by polar trophoblast, and forms a disc of columnar epiblast cells. The blastocyst expands rapidly during the last 24-48 h prior to implantation to 1.7-2.0 mm, and the trophoblast becomes cuboidal with a marked endocytotic apparatus. The morphological evidence, together with previous studies of protein and RNA synthesis, suggests a tooling-up period during early activation with progressive increases in rates of growth and differentiation in the last hours as implantation approaches.     

Estrogen-Dependent Uterine Secretion of Osteopontin Activates Blastocyst Adhesion Competence

Embryo implantation is a highly orchestrated process that involves blastocyst-uterine interactions. This process is confined to a defined interval during gestation referred to as the “window of embryo implantation receptivity”. In mice this receptive period is controlled by ovarian estrogen and involves a coordination of blastocyst adhesion competence and uterine receptivity. Mechanisms coordinating the acquisition of blastocyst adhesion competence and uterine receptivity are largely unknown. Here, we show that ovarian estrogen indirectly regulates blastocyst adhesion competence. Acquisition of blastocyst adhesion competence was attributed to integrin activation (e.g. formation of adhesion complexes) rather than de novo integrin synthesis. Osteopontin (OPN) was identified as an estrogen-dependent uterine endometrial gland secretory factor responsible for activating blastocyst adhesion competence. Increased adhesion complex assembly in OPN-treated blastocysts was mediated through focal adhesion kinase (FAK)- and phosphatidylinositol 3-kinase (PI3K)-dependent signaling pathways. These findings define for the first time specific regulatory components of an estrogen-dependent pathway coordinating blastocyst adhesion competence and uterine receptivity.