Cytochemistry and ultrastructure of the prenatal porcine adrenal medulla

The ultrastructure and cytochemistry of fetal porcine adrenal medullae have been studied at 60, 80, and 100 days of gestation. Adrenal medullae from fetuses at 60 days of pregnancy consisted of norepinephrine cells only. Some cells containing chromaffin granules were seen in the process of mitosis. A few epinephrine cells were present in the outer medullary zone at 80 days at pregnancy, their number increasing by the 100 day of pregnancy. Chromaffin cells containing both norepinephrine and epinephrine storing granules were also present at 80 and 100 days of gestation. Norepinephrine and epinephrine specific granular vesicles in the fetal adrenal medullary cells were smaller than those reported for the adult pig. The general ultrastructural characteristics of the porcine fetal adrenal medulla were similar to those reported for prenatal adrenal medulla of other species.     

Hypothalamic influence on differentiation of the immunoreactive ACTH beta-LPH, alpha- and beta-endorphin containing cells in adenohypophysial primordia of rat fetus in organ culture (author's transl)]

Adenohypophysial primordia were isolated in rat fetuses from day 12.5 to day 15.5 of gestation. The organ culture employed for maintenance of the primordia was made up according to Watanabe et al. (1973). The fixation of primordia in Bouin Hollande's solution was performed after 9, 8, 7 or 6 days of culture when the normal duration of pregnancy was achieved. The cultivated primordia were immunologically studied using different antisera: anti-alpha(17-39)ACTH, anti-beta(1-24)ACTH, anti-beta-LPH, anti-alpha and anti-beta-endorphins, with immunoperoxidase or immunofluorescence techniques, including control experiments of the specificity of the antisera. A similar study was performed on pituitaries removed from normal rat fetuses from day 16.5 of gestation and each day up to birth, and fixated immediately. In vivo the first cells reacting with all the antisera used in this study were observed on day 16.5 of gestation; their number increased during gestation (Fig. 1 A, B and C). Immunoreactive cells with the different antisera could be detected in primordia isolated on day 12.5 of gestation after 9 days of culture. Numerous groups of cells were observed in primordia of older fetuses (Fig. 2 A and B). These data indicate that the corticotropic cells in rat fetuses could start to be differentiated without stimuli from the hypothalamus since primordia were isolated before the appearance of this cell type in normal rat fetuses and before the differentiation of the hypothalamus. The presence of ACTH and other peptides such as beta-LPH or beta-endorphin would support the hypothesis of a common precursor in this cell type existing early in gestation. Similar results were obtained in human fetuses.     

从甲状腺肥大细胞探讨胎儿器官肥大细胞的差异

研究肥大细胞在人胎儿甲状腺发育中数量、分布及组化性质的改变,以探讨胎儿器官发育中肥大细胞的差异。取45例不同胎龄的人胎甲状腺石蜡切片做甲苯胺蓝染色和阿尔辛蓝－－藏红染色,并测定肥大细胞的临界电解质浓度值及进行硫酸小蘖硷荧光染色。结果显示：3月龄胎儿甲状腺内开始出现肥大细胞,数量极少,主要分布在被膜及小叶间结缔组织内,甲苯胺蓝染色肥大细胞颗粒呈淡紫蓝色,阿尔辛蓝－－藏红染色呈蓝色,临界电解质浓度值较低,硫酸小蘖硷染色未见显黄色荧乐的肥大细胞,从3月龄到足月随着胎龄增长,肥大细胞数量缓慢增多,8月龄时肥大细胞经甲苯胺蓝染色,其颗粒呈紫红色,阿尔辛蓝－－藏红染色出现少量含红色和红蓝混合染色颗粒的肥大细胞,临界电解质浓度值偏高,可见少量显黄色荧光的肥大细胞,结果表明：在人胎儿3月龄时甲状腺发育中开始出现肥大细胞,但随胎儿发育肥大细胞的组化性质改变不明显。     

Fine structure of, and ACTH production by, human fetal pituitaries taken at different periods of gestation. An in vitro study

Pituitaries were taken from human fetuses between the 6th and 30th weeks of gestation. Organ and monolayer cultures were prepared. The fine structure of the cultures was examined by electron microscopy and their basal and stimulated ACTH release were studied by radioimmunoassay as a function of time in vitro. It was shown that pituitaries taken from the first trimester of gestation have a capacity of self-differentiation; i.e. there was an increase in the number of cells filled with secretory granules and there was an increase in the number of granules per cell. In contrast, pituitaries taken from older embryos have been losing their secretory granules during the cultivation. We failed to demonstrate any corticotropin responsiveness in pituitary cultures prepared from the 6 to 7-week-old embryos. ACTH release could be stimulated from the 10th week of gestation but in slight measure. Pituitary taken from 16-week-old fetuses revealed an adult-like responsiveness to corticotropin.     

Ontogeny of pituitary responsiveness to corticotropin-releasing hormone in rat

The ontogeny of the pituitary's responsiveness to synthetic rat corticotropin-releasing hormone (CRH) in the late prenatal and early postnatal periods of rats was studied by a superfusion system using whole pituitaries. A significant increase of immunoreactive beta-endorphin (IR-beta-Ep) secretion in response to 10(-10) M CRH but not to 10(-11) M CRH was observed in pituitaries from the 15th day of gestation, the earliest day that we tested, whereas 10(-11) M CRH stimulated IR-beta-Ep release from the pituitaries of 17.5-day-old fetuses. Dose-related IR-beta-Ep secretions induced by 10(-12) M to 10(-10) M CRH were observed in pituitaries of 19.5- and 21.5-day-old fetuses, and 1-, 3- and 9-day-old newborn pups. CRH stimulated not only IR-beta-Ep and IR-adrenocorticotropic hormone (ACTH) but also IR-alpha-melanocyte-stimulating hormone (IR-alpha-MSH) secretions from fetal pituitaries. The content of IR-CRH in the hypothalamic extract from 15-day-old fetus was 6.6 +/- 3.6 pg/hypothalamus (mean +/- S.E.M.) and it gradually increased to reach 212.7 +/- 20.3 pg/hypothalamus on the 21.5th day of gestation. However, the content of IR-CRH in the hypothalamus dramatically decreased just after birth and then rapidly increased again from the 5th day after birth. These data indicate that the responsiveness of corticotrophs to CRH is already present on the 15th day of gestation, when the content of IR-CRH in the hypothalamus is extremely low and that the amount of hypothalamic IR-CRH dramatically dropped for several days just after birth in rats.     

Immunohistochemistry of tonin in rat submandibular gland during development, lactation and secretion

Tonin is a serine protease found in high concentrations in the submandibular gland (SMG) of the adult rat where it has been localized by immunohistochemistry in the granular ducts. The present study examined the development of tonin in the SMG, the effect of lactation and of stimulation of tonin release, using the peroxidase-antiperoxidase technique and antitonin. Tonin-like immunoreactivity first appeared in the primitive striated duct of the SMG on day 19 foetal and increased in intensity as the ducts developed into granular ducts. Reaction product in granules was seen on day 17 postpartum. Its localization within granules was established by immunochemistry of Sepharose beads to which had been coupled the contents of granules isolated from adult rats. The granular ducts of female rats, which are less developed than in the male, showed a marked increase in tonin-immunoreactivity during lactation. Stimulation of tonin secretion by isoprenaline caused massive discharge of tonin-like immunoreactivity into the lumen of the granular ducts during in vitro incubation. However, within one hour complete regranulation was apparent. The secretion was prevented by propranolol. The results indicate that tonin or a tonin-like substance appears in the rat submandibular gland late in gestation in ducts that presumably develop into granular ducts where it is found in abundance in granules in the adult, that the amount in females is increased during lactation, and that most of the granules are discharged during stimulation, only to be rapidly replaced.     

Immunocytochemical study of the GH cells in the anterior pituitary gland of human fetus II. Anencephalic fetus

In order to elucidate the effects of hypothalamic regulation on the morphology of GH cells, light and electron microscopic immunocytochemical examinations were carried out comparing GH cells in the anterior pituitary gland of anencephalic fetus with those of normal fetuses. Three types of GH cells were identified in the anterior pituitary gland of anencephalic fetus as well as in the normal fetus. Type-I is a small, round cell containing a few small secretory granules. Type-III is a large, polygonal cell with numerous large secretory granules. Type-II is a polygonal cell with medium-sized secretory granules. The Type-II GH cell was predominant in both anencephalic and normal fetuses. The most striking difference between anencephalic and normal fetuses was the presence of atypical forms of the Type II cell. These were polygonal cells containing secretory granules, which were either immunopositive or immunonegative to anti-human GH (anti-hGH) serum. Furthermore, two other types of GH cells were identified. The somatomammotroph (SM cell) contained GH and PRL in different granules within the same cell. Also, a different type of the GH cell was noted containing two varieties of secretory granules; one was immunolabeled only with anti-hGH and the other was not immunolabeled to either anti-hGH or anti-human PRL (anti-hPRL). From these results, we suggest that an absence of hypothalamic regulation in the anencehpalic does not seriously modify GH cell morphology but induces an altered GH storage pattern in some of the cells.     

Effects of maternal nutritional status on fetal and placental growth and on fetal urea synthesis in sheep

Fetal and placental growth, and fetal and maternal urea synthesis in late gestation, were studied in 2-year-old Corriedale ewes on a maintenance ration (M) except when subjected to moderate dietary restriction from day 50 to day 100 (RM), day 100 to day 135 (MR) or day 50 to day 135 (RR). In comparison with fetuses of ewes maintained throughout the experiment (MM), RR fetuses were smaller and RM fetuses were larger whereas MR fetuses were unaffected; all restrictions were associated with increased placental size. Fetal urea synthesis at day 133 in the well-nourished ewes (MM) was 21.5 mg N h-1 kg-1 increasing to, respectively, 25.7, 27.3 and 38.8 mg N h-1 kg-1 in groups MR, RM and RR; these values were 1.6, 3.9, 2.2 and 3.8 times the maternal rates of synthesis. On the basis of the observed urea synthesis rates, amino acid oxidation could have accounted for up to, respectively, 32, 38, 40 and 57% of fetal oxygen consumption in groups MM, MR, RM and RR. Amino acids, in addition to their role in tissue accretion, may be key energy substrates for the fetus.     

Gonadal dysgenesis induced by prenatal exposure to ethinyl estradiol in mice

Daily oral administration of ethinyl estradiol (0.02, 0.2, or 2.0 mg/kg of body weight) to pregnant Jc1:ICR mice resulted in ovotestis and intra-abdominal testis with persistent Müllerian duct and Wolffian duct in male fetuses and ovarian hypoplasia in female fetuses when it was given from day 11 through day 17 of gestation (before gonadal differentiation in the fetus). The ovotestis consisted of testicular and ovarian portions. In the testicular portion, a few solid seminiferous tubules containing spermatogonia, some with pachytene nuclei with Sertoli cells and compact interstitial tissue including Leydig cells, were seen. In the ovarian portion, pachytene nuclei were seen. The intra-abdominal testis was smaller and contained more spermatogonia per tubule in cross section than the control testis. These findings suggest that in male fetuses ethinyl estradiol affects Sertoli cell differentiation resulting in suppression of Müllerian inhibiting factor. On the other hand, in the ovarian hypoplasia, the primordial follicles and follicular cells in a primordial follicle were significantly decreased in number, and the number of the degenerated primordial follicles was significantly increased. It seems likely that ethinyl estradiol affects the intimate contact between follicular cells and oocytes to cause degeneration of primordial follicles.     

Possible mechanism of congenital malformations induced by exposure of mouse preimplantation embryos to mitomycin C

ICR mice were treated intraperitoneally with mitomycin C at 5 mg/kg on day 3 of gestation. On day 18 of gestation, fetuses of treated dams were inspected for external, skeletal and visceral malformations. At 6 or 12 hr after mitomycin C treatment, the blastocysts were obtained from the uteri of treated dams and the degenerated cells within inner cell mass (ICM) and trophectoderm (TE) tissues were examined microscopically. On day 5, 8, 11, or 18 of gestation, the uteri of treated dams were obtained and those including embryos/fetuses and placentae were examined histologically. Finally, on each of gestational days 5-14, the blood of the treated dams was collected and the hematological parameters determined. Pre- and postimplantation losses in the dams treated with mitomycin C were significantly increased; increased frequency of abdominal wall defects and lumbar ribs in term fetuses, decreased fetal weight, and increased placental weight were noted as well. No significant increase in visceral malformations was found in term fetuses treated with mitomycin C. Frequency of degenerated cells within ICM and TE of blastocysts from dams treated with mitomycin C was significantly increased as compared with the controls. In dams treated with mitomycin C, decidua developed insufficiently and the trophoblast giant cell layer was not separated from the uterine lumen by maternal components; hemorrhage from the denuded trophoblast giant cell layer into the uterine lumen was noted. The number of erythrocytes, as well as hemoglobin concentration, hematocrit, and the percentage of reticulocytes in blood of dams treated with mitomycin C were significantly lower from days 6-12 of gestation, as compared with controls. The results of the present study showed that an increase in number of degenerated cells within blastocysts results in preimplantation loss and both maternal and embryonic hypoxia during major organogenesis results in postimplantation loss and congenital fetal malformations.     

Bovine nuclear transfer embryo development using cells derived from a cloned fetus.

Many different cell types have been used to generate nuclear transfer embryos and fetuses. However, little is known about the potential of fibroblasts derived from a nuclear transfer fetus as donor cells for nuclear transfer. The ability of cloned fetuses or animals to be cloned themselves is of great interest in determining whether successive generations of clones remain normal or accumulate genetic or phenotypic abnormalities. We generated a bovine fibroblast cell line from a cloned fetus, that continued to divide beyond 120 days (94 doublings,18 passages) in continuous culture. As long-term survival of cells in culture is a desirable characteristic for use in transgenic cell production, passage 2 and 18 cells were compared as donor cells for nuclear transfer (NT). When cells from passage 2 (2 weeks in culture) and passage 18 (4 months in culture) were used for nuclear transfer, there was no significant difference in development rate to blastocyst (35.4 versus 44.6%, P=0.07). A greater proportion of late passage cells were in G0/G1 whether under serum-fed (64 versus 56%, P<0.01) or serum-starved (95 versus 88%, P<0.01) culture conditions. Following embryo transfer, equivalent day 30 pregnancy rates were observed for each group (P 2: 2/19 versus P 18: 2/13). A slightly retarded fetus was surgically removed at day 56 and the remaining three fetuses died in utero by day 60 of gestation. Our results show that fibroblast cells derived from regenerated cloned fetuses are capable of both in vitro and in vivo development. The longevity of this regenerated cell line would allow more time for genetic manipulations and then to identify stable transfected cells prior to their use as NT donor cells. Although no live fetuses were produced in this study the results provide encouraging data to show that a cloned fetus can itself be recloned to produce another identical cloned fetus. Further studies on this and other recloned fetuses are necessary to determine whether the failure to produce live offspring was a result of inadequate sample size or due to the cell type selected.     

Catecholamine-storing cells in the adrenal medulla of the pre- and postnatal rat

Summary The development of the rat adrenal medulla was studied at the ultrastructural level with particular emphasis placed on early discrimination of different catecholamine-storing cells. The first granule-containing cells, phaeochromoblasts, were seen at day 15 of gestation migrating into the anlage of the cortex. These cells were characterized by a few small granules (80–120 nm in diameter) and a high nuclear to cytoplasmic ratio. Presumably due to differentiation into chromaffin cells, they were no longer present after the eighth postnatal day. Maturation of phaeochromoblasts was indicated by an increase in number and size of their storage granules and a decrease in the nuclear to cytoplasmic ratio. Noradrenaline and adrenaline cell types were first clearly discernible at day 21 of gestation. Another cell type, a giant cell, was also recognized at this stage. In the adult animal, noradrenaline, two morphologically different types of adrenaline, and small granule-containing cells were observed.By applying acetylcholinesterase histochemistry, it was found that at day 17 of gestation a small population of granule-storing cells showed strong positive staining in the endoplasmic reticulum. In the adult animal this cell type was further characterized by small-storage granules. Other chromaffin cells began to show weak staining within the endoplasmic reticulum at day 19 of gestation. This staining appeared more frequently within adrenaline than noradrenaline cells. However, even in the adult animal many cells of both types were completely negative.It is concluded that acetylcholinesterase histochemistry is a useful method for early discrimination of small granule-containing cells in the developing rat adrenal medulla.Supported by grants from the Deutsche Forschungsgemeinschaft     

Fetal laser surgery in genetic polydactyly mice

The first digital ray of the hindlimb plate in Slc:ICR mouse fetus was irradiated with 2 watts argon laser beam for 0.3 sec after releasing from the abdominal cavity and envelop of uterine myometrium on day 13 of gestation, and then the fetuses were allowed to develop in the abdominal cavity contacting with the uterus via the placenta exo utero until term. ICR mouse fetuses which received fetal laser surgery lost their first digits completely, resulting in 4-digit hindfoot on day 18 of gestation. The homozygous Polydactyly Nagoya (Pdn/Pdn) mice exhibit 1-3 extra digits both in the fore- and hindlimbs preaxially. The extra digital rays in the left hindlimbs of Pdn/Pdn fetuses were irradiated with 2 watts argon laser beam for 0.3 sec on day 13 of gestation exo utero. The left hindlimbs of the Pdn/Pdn fetuses which received fetal laser surgery lost their preaxial extra digits on day 18 of gestation, resulting in 5 digits, though their 1st digit was triphalangia. The combination of a laser instrument with the fetoscope and/or ultrasound scanner may promote the fetal surgery of congenital anomalies in humans.     

In vivo and in vitro studies on the appearance of LHRH neurons in the hypothalamus of perinatal rats

Summary Ontogenetic development of LHRH-containing neurons was studied by fluorescence and enzyme immunohistochemistry in rats. In in vitro studies, the tissues of the septal-chiasmatic and mediobasal hypothalamic areas of fetal rats on day 16.5 or 18.5 of gestation were trypsinized separately for dissociation of the neural cells, and cultured for several days. Immunopositive reaction against LHRH was first detected in nerve cells derived from both areas of the hypothalamus of the fetuses on days 16.5 and 18.5 of gestation, after 8 and 6 days culture, respectively. The cells were small, and seemed to be bipolar in morphology indicating an axon and arborized dendrites. Immunopositive material occurred in the cell soma as well as in the cellular processes. In in vivo studies, immunopositive material, possibly deposited in nerve fibers, appeared first in OVLT and simultaneously in the external layer of the median eminence of fetuses on day 20.5 of gestation. The immunoreactive fibers increased in number in both parts with development, especially after birth in the median eminence. No immunopositive material was detected within any neural cell bodies nor in the cytoplasm of any ependymal cells.This work was financed by the Ministry of Education, Japan. No. 257008. We would like to thank Dr. Katsuhiko Saito (Department of Surgery, Tokushima University) for his kind advice on the preparation of the antibody used for the immunofluorescence study.     

Developmental changes in proliferative activity of cells of the murine Rathke's pouch

Summary To clarify proliferative activity in the cells of the Rathke's pouch of the rat, we studied the labeling index using bromodeoxyuridine-immunohistochemistry. Rat fetuses were removed 1 h after transplacental injection of bromodeoxyuridine on day 11.5–21.5 of gestation, and were subsequently used to examine cellular proliferation. Although the labeling index within the Rathke's pouch was 30% on day 12.5, it decreased with development and by day 18.5 of gestation had a value of about 5%. The labeling index within Rathke's pouch was not homogeneous throughout the entire pouch, but tended to be higher in regions where cells were more densely packed. This heterogeneous pattern of distribution of labeling index values continued until day 15. On that day, immunoreactive ACTH cells first appeared in the region where the labeling index was low. From day 17 of gestation, the uneven distribution of the labeling index became vague and, simultaneously, the distribution of ACTH cells became homogeneous throughout the pouch. It was concluded that proliferation of the epithelium of Rathke's pouch is heavily involved in the growth of the pouch until at least the appearance of ACTH cells.     

Prostaglandin production by porcine allantochorion in vitro: effect of cortisol infusion in vivo.

The effect of cortisol infusion into the porcine fetus on subsequent prostaglandin (PG) production in vitro by the fetal placenta (the allantochorion) was studied. Also, the possible in vitro effects of glucocorticoids and other steroids on PG production by dispersed cells were examined. Two fetuses in each of 6 sows were catheterized on day 100 or 101 of gestation (normal gestation is 114-116 days); one was infused with cortisol (6 mg/day) and one with saline for 5 days beginning on day 103. On day 108, fetal allantochorionic tissue was aseptically collected from the infused fetuses and 2 uninfused litter mates (controls). Pieces of tissues were cut from the allantochorion (4 sows) and dispersed cell preparations were made from each fetus (4 sows). Each preparation was cultured for 24 h, and the production of PGE2, PGF2 alpha, and 6-keto-PFG1 alpha (prostacyclin metabolite) measured. In vivo cortisol infusion had no significant effect on the in vitro production of PGE2 or PGF2 alpha by tissues or dispersed cell preparations. However, tissue from the fetuses infused with cortisol produced significantly less 6-keto-PGF1 alpha than uninfused controls (54% of control, p < 0.05). The dispersed cells from uninfused fetuses and 2 cortisol-infused animals were also incubated for 24 h with 10(-7) and 10(-9) M concentrations of estrone, estradiol, progesterone, cortisol, and dexamethasone, and the production of PGE2, PGF2 alpha, and 6-keto-PFG1 alpha was measured. No significant effect of any of these steroids in vitro on prostanoid production was observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of estrogen receptors in the efferent ductule of male sheep fetuses during gestation

There is as yet no report about the developmental changes of estrogen receptors (ERs) in the male reproductive system of the sheep fetus. In the present study, the testis, efferent ductule, and epididymis of sheep fetuses were collected at days 70, 90, and 120 of gestation and in the newborn lamb. ER alpha (ERalpha) and ER beta (ERbeta) were detected by immunohistochemistry. The results showed that ERbeta staining was negative in all of the examined tissues throughout gestation, whereas ERalpha immunoreactivity was only located in the nuclei of the efferent ductule epithelium. In addition, both ERalpha staining intensity and the number of ERalpha-positive cells were higher at day 90 of gestation, compared with that at day 70 and at birth. These results suggest that estrogen may play important roles in efferent ductule development in sheep fetuses.     

Ovarian follicular cell hyperplasia in fetal mice treated transplacentally with ethinyl estradiol

The occurrence of follicular cell hyperplasia was studied by light and electron microscopy in fetal mouse ovaries exposed to ethinyl estradiol (EE) from day 11 through day 17 of pregnancy. Pregnant mice were given EE in olive oil (0.02, or 0.2 mg/kg of body weight) and were sacrificed on day 18. The female fetuses were examined for ovarian histogenesis. Follicular cell hyperplasia was detected in both of the experimental groups, but the incidence was statistically significant only in fetuses exposed to 0.2 mg/kg of EE. Light and electron microscopic observations of the ovaries showed that the hyperplasia was located in the medullary region, and the follicular cells showed pleomorphism. Accumulation of abundant lipid droplets, enlarged rough endoplasmic reticulum with granular material, dense bodies, and vague masses of fibrous structures were seen in the cytoplasm. These morphological observations indicate that hyperplasia of follicular cells in fetal mouse ovaries at term can be induced by prenatal treatment with EE.     

Ontogeny of the endocrine pancreas in sea bass (Dicentrarchus labrax): an ultrastructural study. II. The big and secondary islets

The big and secondary islets of sea bass larvae were characterized ultrastructurally from, 25 to 60 days after hatching. From the 25th day, big islets consisted of inner type II and III, external type I and peripheral type IV cells. From the 55th day, type V cells appeared in limited peripheral areas. Secondary islets, first found in 32-day-old larvae, were made up of inner type II and III, external type I, and peripheral either type IV and V cells (type I islets), or only type V cells (type II islets). Type I cells contained secretory granules with a fine granular, low-medium electron-dense material, whereas the secretory granules of type II cells were smaller and had a high electron-dense core with diffused limits; needle and rod-like crystalloid contents were occasionally found. Type III secretory granules posessed a homogeneous, high or medium electron-dense material with or without a clear halo. Type IV cells had secretory granules with a polygonal dense core embedded in a granular matrix and granules containing a high or medium electron-dense material. Type V cells had secretory granules with a fine granular, high or medium electron-dense content. These cell-types correlated with cells previously identified immuno-cytochemically, as regards to their distribution in the islets, and related to those characterized ultrastructurally in adult specimens. Thus, types I, II, III, IV and V correspond to D₁, B, D₂, A and PP cells, respectively. From the 32nd day onwards, endocrine cells of all the different types were found grouped, type V cells also being observed in isolation close to pancreatic ducts and/or blood vessels. Small groups consisting of type I and II cells were found in 40-day-old larvae. A mitotic centroacinar ductular cell containing some secretory granules similar to those of type I cells, was seen adjacent to a type I cell. As the larvae grew older, the endoplasmic reticulum developed, the number of free ribosomes decreased, and the number and size of the secretory granules increased. Dark type I, II, III, IV and V cells were found in the islets and cell clusters from the 55th day onwards.