Endometrial receptivity and embryo implantation in carnivores-commonalities and differences with other mammalian species.

Endometrial receptivity and embryo implantation processes are a major point of pregnancy failure in many mammalian species, including humans. Although reproductive biology in many carnivore species remains enigmatic, the few that have been studied so far are invaluable comparative models. The goals of this review are to (1) summarize current data on the mechanisms involved in uterine receptivity and embryo implantation in carnivores, including commonalities and differences with other mammalian species and (2) identify research priorities to better understand a key phenomenon in a critical group of mammals. Besides unique reproductive traits in some carnivores (induced vs. spontaneous ovulation in cats, ovulation at the germinal vesicle stage in dogs), preimplantation embryo development is comparable with other orders. However, the timing of implantation varies, especially in species having an embryonic diapause. Mechanisms involved in endometrial receptivity and decidualization still remain to be fully understood, but specific markers have already been identified. Importantly, the use of endogenous hormones to control the ovarian activity may impact endometrial receptivity and subsequent embryo implantation. Next, research efforts should take advantage of advanced technologies to further study embryo implantation in carnivores and to provide more relevant models to reproductive medicine or for the conservation of rare and endangered species.

A mechanism for virilization of female spotted hyenas in utero.

Female spotted hyenas exhibit male-like genitalia and dominance over males. Hyena ovarian tissues incubated in vitro produced large quantities of the steroid hormone precursor androstenedione. The activity of aromatase, which converts androstenedione to estrogen, was one-twentieth as great in hyena versus human placental homogenates. In comparison, the activity of 17 beta-hydroxysteroid dehydrogenase, which converts androstenedione to testosterone, was equal in the two homogenates. The limited aromatase activity may allow the hyena placenta to convert high circulating concentrations of androstenedione to testosterone, which results in virilization of the fetal external genitalia and possibly destruction of fetal ovarian follicles. Androstenedione production by residual ovarian stromal cells during reproductive life accounts for the epigenetic transmission of virilization in female spotted hyenas.

Changing glial organization relates to changing fiber order in the developing optic nerve of ferrets.

The structures of the developing eye-stalk and the relationships of early retinofugal fibers as they pass through the stalk, chiasm, and tract have been studied by light and electron microscopical methods in fetal ferrets aged 23-27 days. The early eye-stalk can be divided into two parts: a narrow extracranial part has a narrow lumen and is lined by few cells, whereas a thicker intracranial part has a wider lumen and is lined by several rows of cells. At the earliest stages no axon bundles are recognizable in the stalk, but fibers of the supraoptic commissure are already beginning to cross the midline in the diencephalon. Subsequently, as retinofugal axons invade the stalk, the glia of the extracranial part of the stalk have an interfascicular distribution and axon bundles are separately encircled by glial cytoplasm. In the intracranial part, as in the chiasm and tract, the glial cells occupy a periventricular position and send slender radial cytoplasmic processes to the subpial surface; these pass between groups of axons that here lie immediately deep to the subpial glia. Whereas axonal growth cones have no evident preferred distribution in the extracranial stalk, they tend to accumulate near the pial surface intracranially. The boundary between the two types of organization shifts as development proceeds so that the interfascicular glial structure of the early extracranial stalk first encroaches upon the intracranial parts and later appears in the chiasm. The characteristic adult arrangement of fibers in an age-related order in the optic chiasm and tract, but not in the optic nerve, can be understood if axonal growth cones are guided toward the pial surface by radial glia but not by interfascicular glia. From the distribution of the growth cones, this is what appears to happen.

Early uncrossed component of the developing optic nerve with a short extracerebral course: a light and electron microscopic study of fetal ferrets.

During the study of the developing optic nerve described in the preceding paper (Guillery and Walsh, '87), small bundles of nerve fibers were seen passing between the optic nerve and the ipsilateral hypothalamus of 24-to 27-day-old prenatal ferrets. The bundles appear before any other fiber groups of the retinofugal pathway and are identifiable while the main portions of the retinofugal system are growing into the optic tracts. The bundles, made up of 50 or more axons, leave the optic nerve, emerge through the otherwise continuous layer of subpial glia and through the basal lamina of the nerve, run a short, naked, extracerebral course among collagen fibers and presumed fibroblasts, and then re-enter the central nervous system, passing rostrally and dorsally to the superficial parts of the ipsilateral hypothalamus away from the region of the chiasm. These fibers represent the earliest link between the optic nerve and the brain, but their course is not followed by the majority of retinofugal fibers developing later, which pass toward one or the other optic tract.

Sexual dimorphism of perineal muscles and motoneurons in spotted hyenas.

Female spotted hyenas are known for their male-like genitalia, high levels of aggression, and dominance over males, characteristics which are attributed to exposure to elevated levels of testosterone in utero. Although the nervous system of spotted hyenas has not previously been examined, one might predict that neural systems which are sexually dimorphic in other mammals would be monomorphic in this species. Spinal motoneurons which innervate muscles associated with the phallus are located in Onuf's nucleus and are more numerous in males than in females in a wide array of mammals. Onuf's nucleus was examined in adult and neonatal spotted hyenas and, contrary to expectation, was found to be sexually dimorphic in the typical mammalian pattern: Males have significantly more motoneurons in Onuf's nucleus than do females. This dimorphism was correlated with a previously undescribed dimorphism in the relevant target musculature. Specifically, the morphology of the bulbocavernosus muscle is distinctly different in male and female spotted hyenas. Pregnant hyenas were treated with anti-androgen in an attempt to interfere with the actions of androgen during fetal development. Motoneuron number in Onuf's nucleus and the morphology of the bulbocavernosus muscle were feminized in males exposed to anti-androgen in utero.

Neurotransmitter chemistry of lissencephalic cortex induced in ferrets by fetal treatment with methylazoxymethanol acetate.

Treatment of pregnant ferrets with 15 mg/kg of methylazoxymethanol acetate (MAM) at 33 days of fetal gestation results in offspring with cortical hypoplasia and lissencephally. Neurochemical analysis of 5 areas of cortex from 8-week-old offspring of MAM- or vehicle-treated jills indicated an overall enrichment in markers for catecholaminergic (tyrosine hydroxylase, norepinephrine) and cholinergic (choline acetyltransferase, acetylcholine) terminals but minimal change in the concentration of GABAergic markers (glutamate decarboxylase, gamma-aminobutyric acid); however, there did not appear to be a direct, inverse relationship between the concentration of catecholaminergic and cholinergic markers and the degree of hypoplasia in cortical subareas unlike what has been found previously in the rat.

Placental iron transfer regulation in the haemophagous region of the badger placenta: ultrastructural localization of ferritin in trophoblast and endothelial cells.

The haemophagous region of the badger (Meles meles L.) placenta is involved in iron transfer, and maternal erythrocytes are the only source of iron for the fetus. This ultrastructural study provides morphological evidence of placental ferritin iron storage intervening if haem-iron uptake exceeds fetal needs. Such placental ferritin is assumed to play an active role in iron transfer regulation. In this paper we demonstrate that this regulatory process of iron transfer by storage in placental ferritin occurs on at least two levels: the cytotrophoblast and the fetal endothelium. The latter could then act as a final monitor in the transfer of iron into the fetal circulation.

The story of the spotted hyena and the hermaphrodite: biological order and enigma.

The complex problem of genital masculinization is illustrated in this article from the perspectives of comparative zoology, and pediatric plastic surgery on intersex children. In addition, criticism and recommendations coming from patient internet communications are introduced into the medical community.

Piecing together the puzzle of carnivore reproduction.

Recent advances in feline and canine reproductive studies demonstrate how methodically piecing this information together is beginning to reap rewards for wildlife conservation programs. Non-invasive endocrinology can be used to monitor female reproductive function, time con-specific introductions or AI, and diagnose pregnancy. Sperm morphology characteristics and cell membrane function may be genetically inherited and differ between genetically diverse and inbred species/populations in felids. It is not clear if the same is true for the endangered red wolf. While standards exist for freezing feline and canine sperm, new information using fluorescent staining and zona penetration assays (ZPA) indicates that significant damage can occur during pre-freeze cooling, and may also be related to a species' genetic diversity. Posthumous gamete salvage from genetically valuable animals not only provides a means to study sperm and oocyte physiology but also to assist with genetic management of populations. Using the knowledge gained, IVM/IVF and ICSI have been successful in the domestic cat and AI has resulted in offspring in numerous non-domestic felids. However, understanding the processes of IVM/IVF is still not well understood in canids. New information reveals that sperm and the cumulus cells may be integral to oocyte maturation and that canine epididymal sperm are not capable of undergoing fertilization. The acquisition of knowledge and application of biotechnologies lags behind for non-domestic canid conservation programs.

Embryo technology in conservation efforts for endangered felids.

Most of the 36 species of wild cats are classified as threatened, vulnerable or endangered due to poaching and habitat loss. The important role of assisted reproduction techniques (ART) as part of a multifaceted captive breeding program for selected wild cat species is gradually gaining acceptance. This recognition is a result of the progress made during the last decade in which the feasibility of oocyte recovery from gonadotropin-treated females, in vitro fertilization, embryo cryopreservation and embryo transfer (ET) was demonstrated in the domestic cat (Felis catus). Additionally, embryos have been produced in vitro from oocytes matured in vitro after recovery from ex situ ovaries of both domestic and non-domestic cat species and domestic kittens have been born following transfer of these embryos. In vitro fertilization has been successful in at least one-third of wild cat species and kittens were born after transfer of Indian desert cat (Felis sylvestris ornata) embryos into a domestic cat and con-specific transfer of tiger (Panthera tigris) embryos. The domestic cat is not only a valuable model for development of in vitro techniques but may serve as a recipient of embryos from several species of small wild cats.

Cryopreservation of gametes and embryos of non-domestic species.

Many species of mammals are threatened or endangered. Methods of assisted reproduction that are being used with increasing frequency to produce offspring of domestic animals and humans are often viewed as offering innovative ways to reproduce non-domestic species as well. Uncounted millions of live young of domestic or laboratory species have been produced from gametes and embryos stored at -70 degrees C or below, sometimes for as long as 25 to 35 yrs. Such methods of cryopreservation are now being applied with increasing frequency and urgency to preserve gametes and embryos of non-domestic and threatened species to establish "genome resource banks" or "frozen zoos." But levels of success to produce live young from such cryopreserved gametes or embryos vary considerably from species to species, as well as from individual to individual. It is sometimes thought that differences among species in fundamental characteristics of their gametes may determine the efficacy of cryopreservation and the production of live young. However, it may not be that ineffective cryopreservation is responsible for low success rates. Rather, the limiting factor may be insufficient information and knowledge of the most basic reproductive biology of such non-domestic species. Even standard methods of cryopreservation may be completely adequate to act as a "temporary" expedient to preserve germplasm of non-domestic species to permit time to acquire a fuller understanding of the biology and behavior of non-domestic species.

Noninvasive monitoring of fetal growth and development in the Siberian polecat (Mustela eversmanni).

The Siberian polecat (Mustela eversmanni) is the preferred species to assess procedures and establish normative values for application in the related and endangered black-footed ferret (Mustela nigripes). This study was undertaken to physically, ultrasonographically, and radiographically evaluate fetal development in a spontaneously breeding captive Siberian polecat population. Ultrasonographically, fetal sac enlargement allowed presumptive pregnancy detection as early as 12 days of gestation, the fetal pole was the first definitive sign of pregnancy at about 18 days of gestation, when the fetal heart beat also appeared, and definitive pregnancy detection by ultrasound was essentially 100% accurate after 18 days. The estimation of fetal number by ultrasound was less reliable than by radiography, as it is in other litter-bearing species. Crown-rump growth, organ differentiation, and calcification patterns resembled those of domestic carnivores except that comparable developmental stages in polecats occurred at disproportionately later times, suggesting that young Siberian polecats are delivered in a less developed state. Careful palpation permitted detection of pregnancy after day 17 but with less certainty than with ultrasound. Radiographic evaluation was insensitive and of limited value for pregnancy detection until near term. Litter number and fetal detail were difficult to assess until ossification could be observed, 3-6 days before parturition.

[The preimplantation embryonic development of 2 species of mammals from the family Mustelidae (Mustela erminea and Mustela vison)]. / Doimplantatsionnoe razvitie zarodyshei dvukh vidov mlekopitaiushchikh iz semeistva kunitseobraznykh (Mustela erminea i Mustela vison).

The preimplantation development of common weasel and American mink embryos was studied using light microscopy. Oocytes and blastomeres of these embryos are rich with lipids synthesized during oogenesis. Apparently, most lipids are utilized during the trophoblast formation. Large spherical blastomeres protecting zona pellucida are formed by the stage of implantation. The shape of the blastocyst, as well as central superficial implantation, are typical for carnivores and distinguish the studied order from other ones (e.g., from rodents or artiodactyls). Several aspects of evolution of mammals are discussed. A suggestion is made that differences between orders in the shape of the blastocyst and ways of their implantation reflect poly-phyletic origin of mammals.

[The cytochemical properties of the protective barrier of the duodenum in carnivorous animals in ontogeny]. / Tsitokhimicheskie svoistva zashchitnogo bar'era dvenadtsatiperstnoi kishki plotoiadnykh zhivotnykh v ontogeneze.

Studying the duodenal ontogenesis by means of morphometric and histochemical methods demonstrated that protective barrier of the organ is formed in the embryonic period and may be characterized by chemical composition of its secretion as a resistant barrier, formed by neutral and acid glycoproteins.

Cell production gradients in the developing ferret isocortex.

The accumulation of cells within the cortical plate was studied in ferrets at two developmental ages. A survey method based on the presumed radial organisation of cortical neuron production was used to sample variations in cell production along the rostrocaudal and laterodorsal axes of the brain. The resulting cell counts confirmed the presence of a gradient of cortical plate formation, with a rostrolateral focus. These findings were discussed in relation to some recent teratological studies on brain development in ferrets, where there has been a lack of normative data.

[Late embryonic development of the vomeronasal complex of the cat (Felis silvestris)]. / Zur spätembryonalen Entwicklung des Vomeronasalkomplexes der Katze (Felis silvestris).

An examination of 2 feline embryos in different stages of development (overall length 60 and 115 mm respectively) reveals a well developed vomeronasal complex in each case. Jacobson's Organs embedded within the paraseptal cartilage form long blind tubes at the base of the septum nasi. The cartilage is caudally tub-shaped and embraces rostrally completely the organ over a considerable length. In this manner a long, nearly tunnel-like tube is formed which represents a modified form of the original outer bar and which has not been described so far in cats. It stretches rostro-ventrally across the branching region of the paraseptal cartilage as far as the mouth of Jacobson's Organ. The dorsal branch of the cartilago paraseptalis on the other hand forms a vertically oriented strip which connects to the lamina transversalis anterior. The ductus nasopalatinus passing through the palate is laterally supported by a cartilago ductus nasopalatini which rostrally to the mouth of Jacobson's Organ forms a unified element with the ventral branch of the cartilago paraseptalis. In the case of the younger cat embryo, this cartilago ductus nasopalatini is yet weakly developed. The ductus nasopalatini of the embryos studied are in an amazingly retarded state of development. The ductus, which are blocked in the early stages of the embryonic development during secondary palate formation, form predominantly solid strands of epithelium. By dissolving the cemented epithelium, the ductus are open. But even in the case of the older embryo of the cat, this process is not completed yet. The short duct connecting Jacobson's Organ with the ductus nasopalatinus is also still closed in both embryos. Such cemented sections of epithelium of the younger embryo reveals an interesting relation between the ductus nasopalatinus and the ductus nasolacrimalis which so far has not been pointed out for mammals. From the point of view of phylogenetics, the locally specialized vomeronasal complex of cats exhibits all the criteria of a progressive development of characteristics.

Embryo transfer and embryonic capsules in the bobcat (Lynx rufus).

Bobcats (Lynx rufus) (n=22) were used to test a surgical embryo transfer protocol for wild felines. Five blastocysts were collected 8-14 days post-initial copulation (PIC). Translucent capsule-like structures were recovered at 12 days PIC and are the first report of such a structure in a felid. Endometrial fibrosis was observed in one cat but, in general, post-surgical fibrosis of the uterus did not seem to impede ova or embryo transport. One embryo underwent cryopreservation and this embryo plus two other transferrable embryos were placed in recipient cats during the course of the study. No pregnancies were maintained; but one non-cryopreserved embryo was detected by ultrasound examination at 2 weeks post-transfer. This study provides valuable groundwork for future studies and warrants optimism for continued research in this area.

Development of ferret limb buds in organ culture.

The forelimb bud explants from ferret embryos at days 20, 21, 22, 23 and 24 of gestation were cultured in a Trowell organ culture system. Satisfactory differentiation of the limb bud skeleton was achieved with limbs from embryos explanted at 22 days of gestation (up to 50 somites). Limb buds explanted at 20 and 21 days had poorly differentiated distal segments while those explanted at 23 and 24 days of pregnancy provided less information because chondrogenesis was already under way at the beginning of culture. Thus we conclude that the optimal time for culture of ferret limb buds is at 22 days of gestation. The level of differentiation corresponded to that obtained with mouse limb buds from 11 to 12 day embryos (40-43 somites). The time taken to achieve this level of differentiation in the ferret was 18 days compared with 6 days in the mouse.