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.

Hand development and sequence of ossification in the forelimb of the European shrew Crocidura russula (Soricidae) and comparisons across therian mammals.

Hand development in the European shrew Crocidura russula is described, based on the examination of a cleared and double-stained ontogenetic series and histological sections of a c. 20-day-old embryo and a neonate. In the embryo all carpal elements are still mesenchymal condensations, and there are three more elements than in the adult stage: the 'lunatum', which fuses with the scaphoid around birth; a centrale, which either fuses with another carpal element or just disappears later in ontogeny; and the anlage of an element that later fuses with the radius. Carpal arrangement in the neonate and the adult is the same. In order to compare the relative timing of the onset of ossification in forelimb bones in C. russula with that of other therians, we built up two matrices of events based on two sets of data and used the event-pair method. In the first analysis, ossification of forelimb elements in general was examined, including that of the humerus, radius, ulna, the first carpal and metacarpal to ossify, and the phalanges of the third digit. The second analysis included each carpal, humerus, radius, ulna, the first metacarpal and the first phalanx to ossify. Some characters (= event-pairs) provide synapomorphies for some clades examined. There have been some shifts in the timing of ossification apparently not caused by ecological and/or environmental influences. In two species (Oryctolagus and Myotis), there is a tendency to start the ossification of the carpals relatively earlier than in all other species examined, the sauropsid outgroups included.

The broad ligament: a review of its anatomy and development in different species and hormonal environments.

The broad ligament is a double fold of peritoneum forming a mesentery for the human female genital tract. We investigated the anatomy of the broad ligament in different species and its hormonal regulation to determine if it had a role in gonadal positioning. The medical and veterinary literature was reviewed for descriptions of broad ligament anatomy and development. In addition, four adult female rats were dissected to compare the macroscopic anatomy of the broad ligament with any homologous structures in the male (n = 2). Detailed review was made of human males with persistent Müllerian duct syndrome (PMDS) and of bovine freemartin calves to determine the effect of abnormal hormonal environments on broad ligament development. Human and veterinary texts show variable broad ligament development between species, most being consistent with the size and shape of the uterus and uterine tubes. The broad ligament in adult female rats is a simple peritoneal fold and is homologous with the mesentery of the testis and vas deferens in males. Patients with PMDS and bovine freemartins have a broad ligament with intermediate anatomy. In PMDS the broad ligament is elongated and narrow, and not attached to the pelvic wall. The broad ligament is the mesentery of the genital ducts, and its anatomy varies with the degree of Müllerian duct fusion. The absence of a human male homologue is unusual, as the genital mesentery persists in male rodents. Apparent lack of a male homologue in the human may relate to obliteration of the processus vaginalis. The variable development of the broad ligament in pathological conditions is consistent with a role for steroid hormones in its development.

Nuclear transfer of synchronized african wild cat somatic cells into enucleated domestic cat oocytes.

The African wild cat is one of the smallest wild cats and its future is threatened by hybridization with domestic cats. Nuclear transfer, a valuable tool for retaining genetic variability, offers the possibility of species continuation rather than extinction. The aim of this study was to investigate the ability of somatic cell nuclei of the African wild cat (AWC) to dedifferentiate within domestic cat (DSH) cytoplasts and to support early development after nuclear transplantation. In experiment 1, distributions of AWC and DSH fibroblasts in each cell-cycle phase were assessed by flow cytometry using cells cultured to confluency and disaggregated with pronase, trypsin, or mechanical separation. Trypsin (89.0%) and pronase (93.0%) yielded higher proportions of AWC nuclei in the G0/G1 phase than mechanical separation (82.0%). In contrast, mechanical separation yielded higher percentages of DSH nuclei in the G0/G1 phase (86.6%) than pronase (79.7%) or trypsin (74.2%) treatments. In both species, pronase induced less DNA damage than trypsin. In experiment 2, the effects of serum starvation, culture to confluency, and exposure to roscovitine on the distribution of AWC and DSH fibroblasts in various phases of the cell cycle were determined. Flow cytometry analyses revealed that the dynamics of the cell cycle varied as culture conditions were modified. Specifically, a higher percentage of AWC and DSH nuclei were in the G0/G1 phase after cells were serum starved (83% vs. 96%) than were present in cycling cells (50% vs. 64%), after contact inhibition (61% vs. 88%), or after roscovitine (56% vs. 84%) treatment, respectively. In experiment 3, we evaluated the effects of cell synchronization and oocyte maturation (in vivo vs. in vitro) on the reconstruction and development of AWC-DSH- and DSH-DSH-cloned embryos. The method of cell synchronization did not affect the fusion and cleavage rate because only a slightly higher percentage of fused couplets cleaved when donor nuclei were synchronized by serum starvation (83.0%) than after roscovitine (80.0%) or contact-inhibition (80.0%). The fusion efficiency of in vivo and in vitro matured oocytes used as recipient cytoplasts of AWC donor nuclei (86.6% vs. 85.2%) was similar to the rates obtained with DSH donor nuclei, 83.7% vs. 73.0%, respectively. The only significant effect of source of donor nucleus (AWC vs. DSH) was on the rate of blastocyst formation in vitro. A higher percentage of the embryos derived from AWC nuclei developed to the blastocyst stage than did embryos produced from DSH nuclei, 24.2% vs. 3.3%, respectively (P < 0.05). In experiment 4, the effect of calcium in the fusion medium on induction of oocyte activation and development of AWC-DSH-cloned embryos was determined. The presence of calcium in the fusion medium induced a high incidence of cleavage of DSH oocytes (54.3%), while oocyte cleavage frequency was much lower in the absence of calcium (16.6%). The presence or absence of calcium in the fusion medium did not affect the fusion, cleavage, and blastocyst development of AWC-DSH-cloned embryos. In experiment 5, AWC-DSH-cloned embryos were transferred to the uteri of 11 synchronized domestic cat recipients on Day 6 or 7 after oocyte aspiration. Recipients were assessed by ultrasonography on Day 21 postovulation, but no pregnancies were observed. In the present study, after NT, AWC donor nuclei were able to dedifferentiate in DSH cytoplasts and support high rates of blastocyst development in vitro. Incomplete reprogramming of the differentiated nucleus may be a major constraint to the in vivo developmental potential of the embryos.

Ultrastructure of oocyte maturation, fertilization, and early embryo development in vitro in the Siberian tiger (Panthera tigris altaica).

The application of assisted reproduction techniques to wild cats has been stalled by a lack of basic knowledge of the reproductive biology in these species. In this study, the ultrastructure of Siberian tiger (Panthera tigris altaica) cumulus-oocyte-complexes (COCs), as well as in vitro produced (IVP) zygotes and embryos were investigated, to estimate the normality of the manipulated reproduction processes. Adult female tigers were subjected to a purified porcine pFSH/pLH stimulation treatment followed by oocyte aspiration. According to morphological appearance at the stereomicroscopical level, COCs were classified as mature, immature, or degenerated, and then allocated into the following groups: presumptively immature COCs, which were in vitro matured (IVM-group) before fixation; presumptively mature COCs, which were either fixed after retrieval (pre-IVF-group), following in vitro insemination (IVF-group) or following in vitro insemination and subsequent in vitro culture (IVC-group). All specimens were processed for light and transmission electron microscopy (TEM). Both the IVM- and pre-IVF-group included oocytes in meiotic stages ranging from prophase I to metaphase II, and some prophase I oocytes in the IVM-group were apparently in their growth phase. The IVF-group presented features of presumptive normal fertilization, but aberrations such as polynucleation were also noted. The IVC-group included cleavage stage embryos of which, however, many were polynucleated. In conclusion, the procedures used for stimulation, aspiration, and classification of COCs resulted in retrieval of a heterogeneous population of oocytes which, following IVF and IVC, displayed a high rate of developmental deviations.

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.

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.

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.

Androgens and masculinization of genitalia in the spotted hyaena (Crocuta crocuta). 1. Urogenital morphology and placental androgen production during fetal life.

According to common understanding of sexual differentiation, the formation and development of a penile clitoris in female spotted hyaenas requires the presence of naturally circulating androgens during fetal life. The purpose of the present study was to determine potential source(s) of such fetal androgens by investigating the timing of urogenital development and placental production of androgen during early and mid-gestation. Fetuses determined to be female by molecular techniques (lack of SRY gene) at days 33 and 48 of gestation had undifferentiated gonads, but the clitoris was already 'masculinized' and was generally similar to the phallus of a 50-day-old male fetus. Wolffian and Müllerian ducts terminated at the urogenital sinus in both sexes and a urethra was present along the entire length of the clitoris and penis. The adrenal gland was large and histologically differentiated at 33 days. Steroid gradients across the uterus (a drop in delta 4-androstenedione, with increases in oestrogen and androgen), and high androstenedione in ovarian veins indicated that ovarian androstenedione was metabolized and secreted as testosterone by the placenta throughout gestation. In vitro, whole or homogenized placentae at days 48 and 58 of gestation (110 days total) metabolized radiolabelled androstenedione into testosterone and oestradiol; the specific enzymatic activity of early placental tissues was higher than at later stages. A human placental homogenate had higher aromatase activity but did not produce testosterone unless aromatase was inhibited. Infusion of labelled androstenedione into the uterine arteries of hyaenas demonstrated the conversion of this substrate into testosterone and oestradiol and their secretion into the fetal circulation. Evidently, androgen is produced by the placenta and secreted into the fetal circulation from early in pregnancy when masculinization is first evident, before differentiation of the fetal ovary.

Androgens and masculinization of genitalia in the spotted hyaena (Crocuta crocuta). 2. Effects of prenatal anti-androgens.

Pregnant spotted hyaenas were treated with anti-androgens to interfere with the unusually masculine 'phallic' development that characterizes females of this species. The effects on genital morphology and plasma androgen concentrations of infants were studied during the first 6 months of life. Although there were consistent 'feminizing' effects of prenatal anti-androgen treatment on genital morphology in both sexes, such exposure did not produce males with extreme hypospadia, as it does in other species, nor did it produce females with a 'typical' mammalian clitoris and external vagina. 'Feminization' of males resulted in a penis with the morphological features of the hyaena clitoris, and 'feminization' of females exaggerated the sex differences that are typical of this species. The effects of treatment were present at birth and persisted for at least 6 months. Treatment of pregnant females with flutamide and finasteride also markedly reduced circulating concentrations of testosterone and dihydrotestosterone in maternal plasma during pregnancy. Plasma delta 4-androstenedione was reduced in the female, but not the male, infants of treated mothers, consistent with an epigenetic hypothesis previously advanced to explain hormonal 'masculinization' of females. The present 'feminizing' effects of prenatal anti-androgen treatment are consistent with contemporary understanding of sexual differentiation, which accounts for morphological variation between the sexes in terms of steroids. However, current theory does not account for the basic genital structure of females and the present data suggest that development of the male penis and scrotum, and the female clitoris and pseudoscrotum, in spotted hyaenas may involve both androgen-dependent and androgen-independent components.

Androgens and masculinization of genitalia in the spotted hyaena (Crocuta crocuta). 3. Effects of juvenile gonadectomy.

Studies involving the administration of anti-androgens to spotted hyaenas during fetal development have raised questions concerning the precise contributions of steroids to phallic growth in these animals. If gonadal androgens promote postnatal penile growth in males, the following would be expected: (a) a period of accelerated growth accompanying achievement of puberty, and (b) a marked reduction in adult penile size and density of penile spines after gonadectomy. If a similar androgenic pubertal process stimulates clitoral growth in these highly 'masculinized' hyaenas, parallel observations in females would be expected; however, the role of oestrogens in accounting for female-typical clitoral development would also have to be considered. The results of the present study suggest a limited role, if any for androgenic stimulation of phallic growth. That is, penile growth was greater during the 10 month period preceding puberty, than during an 18-month period that included the traditional increase in pubertal androgens. In addition, pre-pubertal castration had minimal effects on penile length, diameter, or the presence of penile spines. In females, most clitoral growth also occurred before puberty, although pre-pubertal ovariectomy produced significant reductions in clitoral diameter and the elasticity of the urogenital meatus. These feminine characteristics which normally distinguish the female from the male phallus in this species, were partially restored by a brief period of oestrogen administration. Both sexes displayed erections many years after pre-pubertal castration. The results of the present study suggest that postnatal phallic growth is largely independent of gonadal steroids, with oestrogenic facilitation of female-typical clitoral characteristics in spotted hyaenas.

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.

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.

The chronotopic reordering of optic axons.

Retinal ganglion cell axons become reordered as they pass through the chiasmatic region of the optic pathway. Studies in carnivores and rodents show that the fiber order established in the optic tract is a chronological index of their arrival time during development and that the cause of the reordering may relate to the changing glial environment, as well as to the spatial and temporal distribution of proteoglycans within the developing pathway. Primate optic axons become similarly reordered, allowing one to predict a developmental sequence of ganglion cell genesis from fiber position within the mature optic tract. Fiber position within the tract also anticipates the pattern of geniculate innervation, but a prominent exception to this rule is found in the prosimian Galago. The chronotopic reordering is found in every mammalian species that has been examined, including eutherians and metatherians, suggesting that the mechanism producing it is evolutionarily conserved.

Comparative anatomy of the cardiac foramen ovale in cats (Felidae), dogs (Canidae), bears (Ursidae) and hyaenas (Hyaenidae).

The structure of the foramen ovale from 16 species representing 4 carnivore families, the Felidae, Canidae, Ursidae and Hyaenidae, was studied using the scanning electron microscope. The Felidae were represented by 9 domestic cat fetuses (Felis catus), 2 snow leopard neonates (Uncia uncia), an ocelot neonate (Leopardus pardalis), 2 lion neonates (Panthera leo), a panther neonate (Panthera pardus) and 3 tigers (Neofelis tigris), comprising 2 fetuses and a neonate. The Canidae were represented by a golden jackal neonate (Canis aureus), a newborn wolf (Canis lupus), 8 domestic dog fetuses (Canis familiaris), 3 red fox neonates (Vulpes vulpes) and a dhole neonate (Cuon alpinus). The Ursidae were represented by a brown bear neonate (Ursus arctos), a day-old grizzly bear cub (Ursus arctos horribilis), a polar bear neonate (Ursus maritimus), and 2 additional bear fetuses (species unknown). The Hyaenidae were represented by a striped hyaena neonate (Hyaena hyaena). In each species, the foramen ovale, when viewed from the terminal part of the caudal vena cava, had the appearance of a short tunnel. A thin fold of tissue, the developed remains of the embryonic septum primum, extended from the distal end of the caudal vena cava for a variable distance into the lumen of the left atrium and contributed towards the 'tunnel' appearance in all specimens. It constituted a large proportion of the tube, and its distal end was straight-edged. There was fibrous material underlying the endothelium of the flap, the apparent morphology of which suggested that it comprised cardiac muscle.(ABSTRACT TRUNCATED AT 250 WORDS)

[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.

[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.

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.