Development of the Mouse Dermal Adipose Layer Occurs Independently of Subcutaneous Adipose Tissue and Is Marked by Restricted Early Expression of FABP4

The laboratory mouse is a key animal model for studies of adipose biology, metabolism and disease, yet the developmental changes that occur in tissues and cells that become the adipose layer in mouse skin have received little attention. Moreover, the terminology around this adipose body is often confusing, as frequently no distinction is made between adipose tissue within the skin, and so called subcutaneous fat. Here adipocyte development in mouse dorsal skin was investigated from before birth to the end of the first hair follicle growth cycle. Using Oil Red O staining, immunohistochemistry, quantitative RT-PCR and TUNEL staining we confirmed previous observations of a close spatio-temporal link between hair follicle development and the process of adipogenesis. However, unlike previous studies, we observed that the skin adipose layer was created from cells within the lower dermis. By day 16 of embryonic development (e16) the lower dermis was demarcated from the upper dermal layer, and commitment to adipogenesis in the lower dermis was signalled by expression of FABP4, a marker of adipocyte differentiation. In mature mice the skin adipose layer is separated from underlying subcutaneous adipose tissue by the panniculus carnosus. We observed that the skin adipose tissue did not combine or intermix with subcutaneous adipose tissue at any developmental time point. By transplanting skin isolated from e14.5 mice (prior to the start of adipogenesis), under the kidney capsule of adult mice, we showed that skin adipose tissue develops independently and without influence from subcutaneous depots. This study has reinforced the developmental link between hair follicles and skin adipocyte biology. We argue that because skin adipocytes develop from cells within the dermis and independently from subcutaneous adipose tissue, that it is accurately termed dermal adipose tissue and that, in laboratory mice at least, it represents a separate adipose depot.     

Morphological and histological features of the integument of the New Zealand sea lion Phocarctos hookeri

Abstract

Morphological and histological features of the integument of 2 Hooker’s sea lions, obtained dead in February, were examined. In a 3- week-old pup there were 7 rows of mystacial vibrissae, comprising 40 on the left side of the muzzle and 38 on the right. In the skin of the torso the guard hairs were in the quiescent phase of their growth cycle. In a 2½-year-old specimen a moult was imminent; guard hairs in quiescent follicles were about to be displaced by fibres in developing follicles. Histological features of the older sea lion, which may have been age-related, were coarser collagen bundles in the reticular layer of the dermis and a greater number of secondary follicles. In guard hair follicles of both animals the sweat gland duct opened above the sebaceous gland duct.     

Development of dominant follicles and length of ovarian cycles in post-partum dairy cows

The resumption of ovarian activity after normal calvings was studied in 18 lactating Friesian cows. Since, in 17 cows, first post-partum ovulation occurred without overt oestrous behaviour being detected, the resultant cycles were called 'ovarian cycles'. The mean (+/- s.d.) length of the ovarian cycles was 21.0 +/- 8.7 days. The duration of cycles tended to be normal (18-24 days) or long (greater than or equal to 25 days) when the ovulatory dominant follicles were identified before Day 10 post partum; they were consistently short (9-13 days) when dominant follicles identified after Day 20 post partum ovulated. When such follicles were detected between Days 10 and 20 post partum, long, normal and short ovarian cycles were detected. The number of waves of follicular growth with associated dominant follicles observed during the ovarian cycles tended to be related to cycle length; short cycles had 1 dominant follicle, normal cycles predominantly 2, and long cycles mostly 3 dominant follicles. The mean (+/- s.d.) duration of 13 oestrous cycles studied was 23.1 +/- 2.1 days. Of these cycles, 7 had 3 and 6 had 2 dominant follicles. The oestrous cycles with 3 dominant follicles had a mean (+/- s.d.) duration of 24.0 +/- 1.2 days and the respective dominant non-ovulatory follicles reached maximum sizes on Days 8 and 18, respectively; oestrous cycles with 2 dominant follicles were 22.2 +/- 2.6 days in duration, and the dominant non-ovulatory follicle reached maximum size by Day 8. Ovarian follicular development during the first 45 days of pregnancy was characterized by the growth and regression of successive dominant follicles, each lasting 10-12 days. These results show that the first ovarian cycle was predominantly short when the ovulatory dominant follicle was first detected after Day 20 post partum.     

Two stages of juvenile hormone-mediated growth of secondary follicles in Culex pipiens

To investigate the effect of juvenile hormone deprivation on the growth of secondary follicles during the second gonotrophic cycle, female mosquitoes, Culex pipiens L., were allatectomized daily after the first blood meal. Allatectomy on days 1–3 suppressed growth of secondary follicles indicating that juvenile hormone was required for a second gonotrophic cycle. When allatectomy was performed 4 days or more after the first blood meal, secondary follicles grew, indicating the presence of juvenile hormone. However, if mosquitoes were allatectomized before oviposition, only 25% developed a second batch of eggs after a second blood meal.Allatectomies performed 1 and 24 h after oviposition indicated that additional juvenile hormone was released after deposition of the first batch of eggs, and that this second release was needed for secondary follicles to complete previtellogenic growth. Thus, in C. pipiens, secondary follicles undergo two periods of juvenile hormone-mediated growth-one before and one after oviposition.     

Postnatal development of the skin of the marsupial native cat Dasyurus hallucatus

Skin development of the Northern native cat was examined from birth to weaning at 150 days post partum. An outer layer of cells, termed the periderm or epitrichium, is present on the epidermis of the newborn. This layer of cells is not discernible at 7 days post partum. Skin development of the native cat differs from that of the eutherian mammal. The periderm of the eutherian is no longer discernible when the developing hairs first penetrate the epidermis. In the marsupial, this loss of the periderm occurs well before the appearance of follicles. Melanocytes and Langerhans cells are seen at day 23 post partum, follicles at day 30, sebaceous glands at day 59, and sweat glands at day 67. Thus, when the mother first leaves her young in the nest at about days 60 to 70 of lactation, the skin is at a stage of development that will assist the young with thermoregulation. The skin continues to develop throughout lactation and attains an adult appearance by day 150 post partum.     

An ultrasound-aided study of temporal relationships between the patterns of LH/FSH secretion, development of ovulatory-sized antral follicles and formation of corpora lutea in ewes

To characterize the pulsatile secretion of LH and FSH and their relationships with various stages of follicular wave development (follicles growing from 3 to > or =5 mm) and formation of corpora lutea (CL), 6 Western white-faced ewes underwent ovarian ultrasonography and intensive blood sampling (every 12 min for 6 h) each day, for 10 and 8 consecutive days, commencing 1 and 2 d after estrus, respectively. Basal serum concentrations of LH and LH pulse frequency declined, whereas LH pulse duration and FSH pulse frequency increased by Day 7 after ovulation (P<0.05). LH pulse amplitude increased (P<0.05) at the end of the growth phase of the largest ovarian follicles in the first follicular wave of the cycle. The amplitude and duration of LH pulses rose (P<0.05) 1 d after CL detection. Mean and basal serum FSH concentrations increased (P<0.05) on the day of emergence of the second follicular wave, and also at the beginning of the static phase of the largest ovarian follicles in the first follicular wave of the cycle. FSH pulse frequency increased (P<0.05) during the growth phase of emergent follicles in the second follicle wave. The detection of CL was associated with a transient decrease in mean and basal serum concentrations of FSH (P<0.05), and it was followed by a transient decline in FSH pulse frequency (P<0.05). These results indicate that LH secretion during the luteal phase of the sheep estrous cycle reflects primarily the stage of development of the CL, and only a rise in LH pulse amplitude may be linked to the end of the growth phase of the largest follicles of waves. Increases in mean and basal serum concentrations of FSH are tightly coupled with the days of follicular wave emergence, and they also coincide with the end of the growth phase of the largest follicles in a previous wave, but FSH pulse frequency increases during the follicle growth phase, especially at mid-cycle.     

Artificial induction of superfetation in the European hare (<Emphasis Type="Italic">Lepus europaeus</Emphasis>)

The aim of this study was to induce superfetation in hares. On day 37 or 39 of pregnancy, female hares were treated with GnRH analogue and artificially inseminated with fresh semen. Ultrasonographic examination showed that pregnant females had follicles on the ovary on 37 days of pregnancy. After a few days from insemination performed during pregnancy, all females gave birth to healthy young, and one of the females inseminated on day 37 of pregnancy gave birth to two healthy young 42 days after insemination performed during pregnancy (39 days post first delivery). The same female in the next year gave birth to two healthy young 43 days after second insemination that was performed on the day 37 during pregnancy. The obtained results confirm that superfetation is possible to occur in hares.     

Histological studies on the development of porcine tonsils after birth

Tonsils form the topographically first immune barrier of an organism against the invasion of pathogens. We used histology to study the development of tonsils of pigs after birth. At birth, the tonsils consist of diffuse lymphoid tissue without any lymphoid follicle aggregations. At the age of 7 days, lymphoid follicles appeared in the soft palate tonsil. The lymphoid layer of the nasopharyngeal tonsil, soft palate tonsil, and lingual tonsil became thicker, and lymphoid follicles in the lamina propria were clearly visible at the age of 21 days. Secondary lymphoid follicles were present in the nasopharyngeal tonsil at the age of 50 days, and in the soft palate tonsil at the age of 120 days. Dendritic cells (DCs), CD3⁺ T cells and IgA⁺ B cells in the soft palate tonsil, nasopharyngeal tonsil and lingual tonsil increased continuously, especially during the first 21 days. The results suggested that tonsils have an important role in local immune defense against invading antigens after birth and will be beneficial for understanding the mechanisms of immunity in these animals after nasal and oral vaccination.     

Pattern of follicular growth and resumption of ovarian activity in post-partum beef suckler cows

The ovaries of 18 post-partum beef suckler cows were examined daily, using ultrasound, from Day 5 post partum until a normal oestrous cycle was completed. Periods of growth and regression of medium-sized (5-9 mm) follicles were identified before one medium follicle became dominant (single large follicle greater than or equal to 10 mm). The mean (+/- s.e.m.) number of days from parturition to detection of the first post-partum dominant follicle was 10.2 +/- 0.5. The first post-partum dominant follicle ovulated in 2/18 (11%) cows. The interval from calving to first ovulation (mean +/- s.e.m. = 35.9 +/- 3.3 days) was characterized by the growth and regression of a variable number (mean = 3.2 +/- 0.2; range 1-6) of dominant follicles. The maximum diameter of the dominant follicle increased as the cows approached first ovulation (P less than 0.05). Behavioural oestrus was not detected in 16/18 (89%) cows at first ovulation. Following first ovulation, the length of the subsequent cycle was short (mean = 9.7 +/- 0.5 days; range 8-15 days) in 14/18 (78%) cows and was characterized by the development and ovulation of a single dominant follicle. During oestrous cycles of normal length (mean = 20.6 +/- 0.5 days; range 18-23 days) one (N = 2), two (N = 7) or three (N = 8) dominant follicles were identified. The growth rate, maximum diameter or persistence of non-ovulatory dominant follicles before first ovulation or during oestrous cycles were not different (P greater than 0.05). These data show that, in beef suckler cows, follicular development and formation of a dominant follicle occur early after parturition and the incidence of ovulation of the first dominant follicle is low. The number of dominant follicles that develop before first ovulation is variable; first ovulation is rarely associated with oestrus and short cycles are common after first ovulation. It is concluded that prolonged anoestrus in post-partum beef suckler cows is due to lack of ovulation of a dominant follicle rather than delayed development of dominant follicles.     

Epidermis Is the Site of Action of Tabby (TA) in the Mouse

The site of action of the sex-linked tabby (Ta) locus was analyzed by the technique of dermal-epidermal recombination grafting. Skin components from normal and tabby 14-day embryos were separated, recombined and grown 21 days in testes of histocompatible mice. Grafts of the combinations normal epidermis-normal dermis and normal epidermis-tabby dermis produced predominantly zig-zag hairs. Grafts of the combination tabby epidermis-normal dermis and tabby epidermis-tabby dermis produced hairs with a morphology similar to hairs found in tabby mice. We conclude from these results that the tabby locus acts within the epidermis, and has no effect on the dermis.     

Ultrasound and endocrine evaluation of the ovarian response to PGF2alpha given at different stages of the luteal phase in ewes

The purpose of this study was to evaluate the ovarian response of ewes to two treatments with PGF2alpha using transrectal ovarian ultrasonography and hormone measurements. Fifteen milligrams of PGF2alpha was given to six cyclic Western White Face (WWF) ewes early in the estrous cycle (Days 4 to 7) and to six late in the cycle (Days 10 to 12 after ovulation), and a second treatment was given 9 days after the first. Ultrasound scanning and blood sampling started 7 days prior to the first PGF2alpha treatment and ended 10 days (scanning) or 19 days (blood sampling) after the second PGF2alpha treatment, for both groups of ewes. Mean ovulation rate (2.6 +/- 0.7) did not differ significantly between the ewes first treated early or late in the cycle, or after the first or second treatments with PGF2alpha. The time from treatment to ovulation was longer in ewes first treated early (4.0 +/- 0.3 days) compared to late (2.8 +/- 0.4 days) in the cycle (P < 0.05). Both the number of ovulations (range: 0-7) and time from treatment to ovulation (range: 1-9 days) were highly variable. This variability appeared to be due to the extension of the life span of ovulating follicles that emerged prior to PGF2alpha administration and also ovulation of some follicles that emerged after treatment. When results for first and second treatments were pooled, the total number of follicles > 5 mm in diameter on the day of treatment that failed to ovulate in response to PGF2alpha was higher in ewes first treated early (0.8 +/- 0.2/ewe) compared to late (0.3 +/- 0.2/ewe) in the cycle (P < 0.05). The proportion of detected luteal structures relative to the number of ovulations was lower in ewes first treated early compared to late in the cycle (60 and 86%, respectively; P < 0.05). Disruption of ovulatory follicle dynamics and normal luteogenesis, and variability in the timing of ovulation after PGF2alpha treatments could all contribute to poor or variable fertility when prostaglandins are used for estrus synchronization.     

A histological study on the skin and hairs of PC (poor coat) mice

Light microscopic examinations were done on the skin and hairs of PC (poor coat) mice, maintained as an inbred strain at the National Institute of Health, Japan. The structures of the epidermis, dermis, hair root sheath and the sebaceous glands were normal. Hair bulbs and hair papillae were poorly developed at anagen stage of hair cycle. Having scanty medulla, the hairs were thin and short. The hair cuticle appeared normal. These findings suggest that the defective hair growth in PC mice is caused by deficiencies in cell differentiation and/or proliferation in the hair matrix.     

Regional specification of cutaneous appendages in mammals

Summary The problem of the regional specification of snout vibrissae and dorsal pelage hairs has been analysed in mouse embryos. Reconstituted homo-and heterotopic skin explants, consisting of epidermis and dermis from both regions, were cultured on the chorioallantoic membrane of the chick embryo.Recombinants of 12.5-day upper lip dermis and 12.5-day dorsal epidermis developed a small number of large vibrissal type follicles arranged in a recognizable rectangular vibrissal pattern. The reverse combinations of 12.5- or 14.5-day dorsal dermis and 11- to 12.5-day upper lip epidermis formed a single population of numerous and small follicles arranged in a typical pelage hair pattern (trio groups) or gave rise to a mixed population of follicles with both whiskers and pelage hairs.It is concluded that the dermis is responsible for the regional specification of the cutaneous appendages and their distribution pattern. However, at the time it was isolated, the upper lip epidermis already possesses the information for the morphogenesis of vibrissae, but remains malleable and responsive to the dermal influence.This work was supported in part by DGRST and CNRS     

Synchronization rate, size of the ovulatory follicle, and pregnancy rate after synchronization of ovulation beginning on different days of the estrous cycle in lactating dairy cows

Recently a protocol was developed that precisely synchronizes the time of ovulation in lactating dairy cows (Ovsynch; GnRH-7d-PGF2 alpha-2d-GnRH). We evaluated whether initiation of Ovsynch on different days of the estrous cycle altered the effectiveness of this protocol. The percentage of cows (n = 156) ovulating to the first GnRH was 64% and varied (P < 0.01) by stage of estrous cycle. Treatment with PGF2 alpha was effective, with 93% of cows having low progesterone at second GnRH. The overall percentage of cows that ovulated after second GnRH (synchronization rate) was 87% and varied by response to first GnRH (92% if ovulation to first GnRH vs 79% if no ovulation; P < 0.05). There were 6% of cows that ovulated before the second injection of GnRH and 7% with no detectable ovulation by 48 h after second GnRH. Maximal diameter of the ovulatory follicle varied by stage of estrous cycle, with cows in which Ovsynch was initiated at midcycle having the smallest follicles. In addition, milk production and serum progesterone concentration on the day of PGF2 alpha affected (P < 0.05) size of the ovulatory follicle. Using these results we analyzed pregnancy rate at Days 28 and 98 after AI for cows (n = 404) in which Ovsynch was initiated on known days of the estrous cycle. Pregnancy rate was lower for cows expected to ovulate larger follicles than those expected to ovulate smaller follicles (P < 0.05; 32 vs 42%). Thus, although overall synchronization rate with Ovsynch was above 85%, there were clear differences in response according to day of protocol initiation. Cows in which Ovsynch was initiated near midcycle had smaller ovulatory follicles and greater pregnancy rates.     

Dermal white adipose tissue: a new component of the thermogenic response

Recent literature suggests that the layer of adipocytes embedded in the skin below the dermis is far from being an inert spacer material. Instead, this layer of dermal white adipose tissue (dWAT) is a regulated lipid layer that comprises a crucial environmental defense. Among all the classes of biological molecules, lipids have the lowest thermal conductance and highest insulation potential. This property can be exploited by mammals to reduce heat loss, suppress brown adipose tissue activation, reduce the activation of thermogenic programs, and increase metabolic efficiency. Furthermore, this layer responds to bacterial challenge to provide a physical barrier and antimicrobial disinfection, and its expansion supports the growth of hair follicles and regenerating skin. In sum, this dWAT layer is a key defensive player with remarkable potential for modifying systemic metabolism, immune function, and physiology. In this review, we discuss the key literature illustrating the properties of this recently recognized adipose depot.     

Follicular epithelium of the ovary in the prenatal and early postnatal periods of ontogenesis of the Wrangell Island lemming Dicrostonyx torquatus Pall

In 69 representatives of the Arctic lemming the ovaries have been studied. Morphologucal peculiarities of prefollicular and follicular cells have been revealed during various periods of folliculogenesis, as well as changes in the volumetric density arrangement of cellular organells in the process of their differentiation. It is supposed that during the period preceding formation of the follicles (15.5-20 days of embryonal development and the first three days after birth) the prefollicular cells possess several functions (participation in regulating spatial organization of ovogonia, fagocytosis), the secretory one including. During formation of primordial follicles (1-3 days after birth) the follicular epithelium cells become flatten and their structures orient towards supporting-mechanical function. The following morphological changes of folliculocytes are connected with further growth of follicles, granulosa formation, secretion of follicular fluid in cavitary follicles that form at the beginning of the third week. A coinside in time is noted in formation of primordial follicles and in beginning of their growth in laboratory rodents and in the lemmings, nevertheless, transformation of the growing follicles into the cavitary ones occurs more quickly in the lemming.     

The hair follicle: a paradoxical androgen target organ

Androgens are the main regulator of normal human hair growth. After puberty, they promote transformation of vellus follicles, producing tiny, unpigmented hairs, to terminal ones, forming larger pigmented hairs, in many areas, e.g. the axilla. However, they have no apparent effect on the eyelashes, but can cause the opposite transformation on the scalp leading to the replacement of terminal hairs by vellus ones and the gradual onset of androgenetic alopecia. This paradox appears to be an unique hormonal effect. Hair follicles are mainly epithelial tissues, continuous with the epidermis, which project into the dermis. A mesenchyme-derived dermal papilla enclosed within the hair bulb at the base controls many aspects of follicle function. In the current hypothesis for androgen regulation, the dermal papilla is also considered the main site of androgen action with androgens from the blood binding to receptors in dermal papilla cells of androgen-sensitive follicles and causing an alteration of their production of paracrine factors for target cells e.g. keratinocytes. Studies of cultured dermal papilla cells from sites with different responses to androgens in vivo have confirmed the paradoxical responses. All dermal papilla cells from androgen-sensitive sites contain low capacity, high affinity androgen receptors. However, only some cells formed 5alpha-dihydrotestosterone, e.g. beard but not axillary cells, in line with hair growth in 5alpha-reductase deficiency. Incubation with androgens also stimulated the mitogenic capacity of beard cell media, but inhibited that produced by scalp cells. This suggests that the paradoxical differences are due to differential gene expression within hair follicles, presumably caused during embryogenesis.     

Compensatory ovulatory mechanisms operative after first ovulation in rats unilaterally ovariectomized prepubertally

Ovarian steroid contents and serum concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and prolactin were measured during the days after first ovulation in rats unilaterally ovariectomized in late prepuberty. In addition, follicle counts were made at second estrus and second metestrus. During the cycle following first ovulation, ovarian estradiol contents in unilaterally ovariectomized (ULO) rats were significantly increased as compared to intact rats on the day of metestrus, on diestrus 1 and on second estrus. Ovarian progesterone was significantly increased on the days of metestrus, on diestrus 1, second proestrus and second estrus, but no differences were seen in ovarian androgen contents. After ULO there was an indication of an augmented FSH surge at the first and the second ovulation. Follicle counts revealed that the total number of healthy as well as of atretic antral follicles on the day of second estrus was significantly increased after ULO, due to increased numbers of the smallest antral follicles. At second metestrus the number of larger antral follicles (350-500 micron 3) and the total number of healthy antral follicles was higher after ULO. It is concluded that the compensatory process after ULO involved increased recruitment of small antral follicles. Activities in the remaining ovary were not simply doubled but a new hormonal balance was established.     

Delayed epidermal permeability barrier formation and hair follicle aberrations in Inv-Cldn6 mice

Homozygous mice overexpressing Claudin-6 (Cldn6) exhibit a perturbation in the epidermal differentiation program leading to a defective epidermal permeability barrier (EPB) and dehydration induced death ensuing within 48 h of birth [Turksen, K., Troy, T.C., 2002. Permeability barrier dysfunction in transgenic mice overexpressing claudin 6. Development 129, 1775-1784]. Their heterozygous counterparts are also born with an incomplete EPB; however, barrier formation continues after birth and normal hydration levels are achieved by postnatal day 12 allowing survival into adulthood. Heterozygous Inv-Cldn6 mice exhibit a distinct coat phenotype and histological analysis shows mild epidermal hyperkeratosis. Expression of K5 and K14 is aberrant, extending beyond the basal layer into the suprabasal layer where they are not co-localized suggesting that their expression is uncoupled. There is also atypical K17 and patchy K15 expression in the basal layer with no K6 expression in the interfollicular epidermis; together with marked changes in late differentiation markers (e.g. profilaggrin/filaggrin, loricrin, transglutaminase 3) indicating that the normal epidermal differentiation program is modified. The expression compartment of various Cldns is also perturbed although overall protein levels remained comparable. Most notably induction of Cldn5 and Cldn8 was observed in the Inv-Cldn6 epidermis. Heterozygous Inv-Cldn6 animals also exhibit subtle alterations in the differentiation program of the hair follicle including a shorter anagen phase, and altered hair type distribution and length compared to the wild type; the approximately 20% increase in zig-zag hair fibers at the expense of guard hairs and the approximately 30% shorter guard hairs contribute to coat abnormalities in the heterozygous mice. In addition, the transgenic hair follicles exhibit a decreased expression of K15 as well as some hair-specific keratins and express Cldn5 and Cldn18, which are not detectable in the wild type. These data indicate that Cldn6 plays a role in the differentiation processes of the epidermis and hair follicle and supports the notion of a link between Cldn regulation and EPB assembly/maintenance as well as the hair cycle.     

Ultrastructural analysis between fetal and adult wound healing process of marsupial opossum skin

The opossum delivers a newborn baby equivalent to tremature fetus state by postpregnancy. The peculiarity is advantageous for studies of fetus, because operations to take out fetus from the uterus of a mother are not necessary. When mammalian skin is wounded by full-thickness excision, fetal and adult wound healing processes differ. Fetal-type wound healing does not leave a scar. However, studies of how the fetal wound healing process differs in detail from the adult type are not advanced. We first observed the normal skin development of the gray short-tailed opossum (Monodelphis domestica) using an electron microscope. As for normal skin, an epidermis became multi-layered, and thickened from birth through to 7 days after birth. The quantity of extracellular matrix of the dermis increased thereafter, and several types of cells were found in the dermis. To examine the wound healing, we used material from a 1 day-old newborn baby, and from another 15 days after birth, and compared the wound healing style morphologically. Differences in the constitution of cells and fine structures of the skin were observed, it was obviously suggested that change in the wound healing style from fetal-type to adult-type occurred between 1 to 15 days after birth.