Body measurements correlation and x-ray imaging of three Hystrix javanica fetuses in different fetal development stages.

Hystrix javanica is endemic species in Indonesia. Study about fetal development of Hystrix javanica are very rare because of sample limitation. This study was carried out to describe the morphometrics and x-ray analysis of three fetuses in different stage to give basic information about fetal development of Hystrix javanica. Three fetus samples fixed in Bouin's solution was used in this study. Observation was carried out to identify the characteristic of three fetus samples. This included the pattern of hair, body measurements, body volume, and body weight. X-ray analysis was carried out to know the ossification process in the fetal development. Statistical analysis was carried out using Microsoft 365® Excel program software. Three fetus samples had different specific hair pattern, that was hairless, smooth hairs, and smooth hairs with dense-non dense pattern. Body volume of 1st, 2nd, and 3rd fetus were 23mL, 90mL, and 170mL, respectively. Body weight of 1st, 2nd, and 3rd fetus were 19.5g, 79.22g, and 153.18g, respectively. Pearson's correlation analysis shown strong relationship between total body length, front body length, back body length, horizontal body diameter, vertical body diameter, head length, and head diameter against body volume and body weight of three fetuses. Significant positive correlation was shown between horizontal body diameter, vertical body diameter, and head diameter against body volume and body length with P value < 0.05. Faint radiopaque images showed in the 2nd fetus sample and strong radiopaque images showed in the 3rd fetus sample. Radiopaque images were identified in the teeth, cranium, vertebrae, and extremities bones. In this study we concluded that there was a specific hair pattern in different fetal stage. All body measurements have positive correlation with body volume and body weight and x-ray analysis shown that the ossification of the bone was started to happen while the smooth hair was growth.

Dried bovine placenta improves spermatozoa count in a rat model of male reproductive aging.

BACKGROUND AND AIM: In the male reproductive system, the aging process can lead to infertility. Recently, placenta and its derivatives have been researched as regenerative agents. This study aimed to describe the basic components of dried bovine placenta powder and its potential effects as a regenerative agent in a rat model of male reproductive aging with D-galactose induction. MATERIALS AND METHODS: We divided 15 male Wistar rats, 2 months of age, into three groups: A, the health control group; B, the D-galactose induction group, and C, the D-galactose induction and 10% dried bovine placenta supplementation group. We measured epididymal sperm concentration and testicular weight and volume and analyzed these using one-way analysis of variance. RESULTS: Dried bovine placenta was rich in nutrients, with 61.98% protein, 21.25±2.07 carbohydrates, 8.58% water, 4.93% ash, and 3.27% fat. The mean epididymal spermatozoa concentration of the rats in Groups A, B, and C was 3026×106/mL, 1492.8×106/mL, and 2732.5×106/mL, respectively. The average total testicle weights were 2.44 g, 2.72 g, and 2.57 g, respectively. The average total testicle volumes were 2.29 cm3, 2.49 cm3, and 2.33cm3, respectively. CONCLUSION: Dried bovine placenta powder is rich in nutrients, especially protein. Supplementation with dried bovine placenta can improve epididymal spermatozoa concentration that is important in fertility.

Callus Formation in Fractured Femur of Rats Treated with Injection of Human Umbilical Cord Mesenchymal Stem Cell-Conditioned Medium.

Mesenchymal stem cells-conditioned medium (MSC-CM) is the extraction from stem cell medium containing biological substances, including growth factors and cytokines. These substances play roles in the various functions of body regulatory, including bone formation. However, the effect of MSC-CM derived from human umbilical cord injection in femur fracture healing of rats has not been reported previously. This study aims to see the effect of MSC-CM derived from human umbilical cord injection on the callus formation of bone fracture healing in Wistar rats (Rattus norvegicus). A femur fracture in 54 Wistar rats was made by surgery according to the procedure under sterile conditions. After the surgery, rats were divided into 2 groups of 27, respectively. Injection in the control (0.1 mL/kg body weight NaCl) and MSC-CM group (0.1 mL/kg body weight MSC-CM) was performed on weeks 0, 1, 2, 3, 4, 5, 6, 7, and 8 after surgery. Radiographic images and the femur bone samples were taken and collected on days 1, 7, 14, 21, 28, 35, and 60 after surgery. Bone samples were then fixed in Bouin solution. Histologic preparations were done by the paraffin method, by cutting the tissue blocks into 5 µm thickness and then staining with Mallory aniline blue staining. The results were analyzed descriptively and quantitatively. The result showed that the soft callus formation occurred rapidly and got wider in the MSC-CM group than that of the control group. The administration of MSC-CM injection postfracture surgery to femur fracture cases in rats was capable to accelerate the callus formation.

Secretome improves testosterone and androgen-binding protein production in testicular dysfunction rats induced by cisplatin.

OBJECTIVE: This study was determined to see the effects of secretome on cellular production of testosterone and androgen-binding protein (ABP) using immunohistochemistry in testicle dysfunction due to cisplatin. MATERIALS AND METHODS: Forty-eight rats were divided into four groups: the healthy group, the group with testicular dysfunction, the secretome-treated group with 0.2 ml/kg body weight (BW), and the secretome-treated group with 0.3 ml/kg BW. The immunohistochemistry staining method is used to find out testosterone and ABP reactivity in tissue organs. RESULTS: Very strong testosterone and ABP immunoreactivity were found in Leydig cells of normal testes. While in the Leydig cells of cisplatin-induced testicles, testosterone and ABP immunoreactivity were not observed. Testosterone and ABP were observed 1 week after the second secretome injection. The number of testosterone-immunoreactive cells in the low dose group from week 1 to 3 was 0, 19, and 32, respectively. From week 1 until week 4, the high dose group was 0, 29, 33, and 65, respectively. The number of ABP-immunoreactive cells from the first week until the third week in the low dose group was 0, 28, and 34, respectively. The high dose group from the first week until the fourth week was 0, 26, 58, and 83, respectively. The number of cells that produce testosterone and ABP increased from week 2 to 4. CONCLUSION: The administration of secretome could increase the number of immunoreactive cells toward testosterone and ABP in testicular dysfunction.

An immunohistochemical study of endocrine cells in the digestive tract of Varanus salvator (Reptile: Varanidae).

AIM: The aim of the study was to identify the distribution pattern and frequency of endocrine cell types in the digestive tract of Varanus salvator. MATERIALS AND METHODS: The presence of endocrine cells (glucagon, somatostatin, and serotonin) in the digestive tract (esophagus, stomach, and intestine) was detected using the avidin-biotin complex (ABC) method. RESULTS: Three types of endocrine cells immunoreactive to antisera glucagon, serotonin, and somatostatin were found in the caudal portion of the small and large intestines but were not observed in the esophagus, stomach, and caput and medial sections of the small intestine. Endocrine cells distributed in the digestive tract of V. salvator vary in color intensity, from weak to sharp, in response to the primer antibody. CONCLUSION: Endocrine cells in the digestive tract that is immunoreactive to glucagon, somatostatin, and serotonin are those found in the caudal portion of the small and large intestines. They are varied in distribution pattern, frequency, and color intensity.

Immunolocalization of IL-6 and IL-10 in the testicular tissue of testicular dysfunction rat treated with secretome.

OBJECTIVE: This study was carried out to find out the immunolocalization of Interleukin 6 (IL-6) and Interleukin 10 (IL-10) in the testicular tissue of testicular dysfunction rat treated with secretome from human umbilical stem cells. MATERIALS AND METHODS: Rats were induced with cisplatin for testicular dysfunction condition. After that, the rats were grouped into two categories and were treated with secretome at 0.2 and 0.5 ml/kg BW once every week for 4 weeks. One week later, after the secretome treatment, the rats were sacrificed for histological evaluation using the immunohistochemical method. The preparation slides were examined using a light microscope and were analyzed descriptively and quantitatively. RESULTS: There were no IL-6 and IL-10 immunoreactivities seen in the testicular tissue after cisplatin induction. However, the immunoreactivities of IL-6 and IL-10 were detected after secretome treatment, with both dosages of 0.2 and 0.5 ml/kg BW. These immunoreactivities were detected in the spermatogonia, spermatid/luminal tissue of seminiferous tubule, spermatogenic cells, and Leydig cells. In the cell calculation, the numbers of IL-6 immunoreactive cells were higher at the lower secretome dosage. In contrast, the numbers of IL-10 immunoreactive cells were higher at the higher secretome dosage. CONCLUSION: IL-6 and IL-10 can be localized in the testicular tissue of testicular dysfunction rat after secretome treatment.

An immunohistochemical study on testicular steroidogenesis in the Sunda porcupine (Hystrix javanica).

In the testes of the Sunda porcupine (Hystrix javanica), the expression of the steroidogenic acute regulatory protein (StAR) and steroidogenic enzymes, such as cytochrome P450 side chain cleavage (P450scc), 3ß-hydroxysteroid dehydrogenase (3ß-HSD), cytochrome P450 17α-hydroxylase (P450c17) and cytochrome P450 aromatase (P450arom), was immunohistochemically examined to clarify the location of steroidogenesis. In this study, complete spermatogenesis (spermiogenesis) was observed in the testes of the examined Sunda porcupine, and spermatozoa of the Sunda porcupine had a spatulate sperm head unlike that of rats and mice which has an apical hook. On immunostaining of StAR, P450scc, 3ß-HSD, P450c17 and P450arom, immunoreactivity for all proteins was only detected in the Leydig cells and not observed within the seminiferous tubules, suggesting that the Leydig cells can synthesize both androgen and estrogen from cholesterol in the Sunda porcupine testes.

Effects of secretome on cisplatin-induced testicular dysfunction in rats.

BACKGROUND: Testicular dysfunction is a degenerative disorder characterized by failure in the synthesis of reproductive hormones and spermatogenesis. Secretome derived from the human umbilical mesenchymal stem cell (MSC) has been reported to repair some degenerative disorders. AIM: This study aimed to investigate the effect of secretome derived from the human umbilical MSCs on cisplatin-induced testicular dysfunction in rats. MATERIALS AND METHODS: Thirty-six male Wistar rats were divided into the control and secretome-treated groups. In the secretome-treated group, testicular dysfunction was induced by 3 mg/kg BW of cisplatin intraperitoneally 3 times with 3-day intervals. The secretome-treated group was divided according to dose: Low-dose (0.2 mL/kg BW) and high-dose (0.5 mL/kg BW) groups. Secretomes were injected intraperitoneally once a week for 3 weeks. 1 week after the injection of secretome, the cauda epididymis of the rats was removed for spermatozoa evaluation and histological examination. RESULT: After the injection of secretome, the sperm motility of the high-dose group showed thin wave-like, rare, and slow movements. No abnormal sperm morphology was observed in all the treated groups. The number of spermatozoa increased gradually in the high-dose group after the injection of secretome. The developmental stages of the spermatogenic cells were complete in both spermatozoa groups after the injection of secretome. However, the spermatozoa in the seminiferous tubules of the high-dose group were denser. Vimentin and cytokeratin immunoreactivities were very strong in the high-dose group 1 week after the second secretome injection. CONCLUSION: High-dose secretome derived from the human fetal umbilical cord could increase the number and motility of sperms in rats with cisplatin-induced testicular dysfunction. The administration of high-dose secretome was effective 1 week after the second dose, as indicated by very strong immunoreactivity for vimentin and cytokeratin. Moreover, secretome could promote the regeneration of the seminiferous tubules of both the groups.

The types of endocrine cells in the pancreas of Sunda porcupine (Hystrix javanica).

AIM: To identify the types of endocrine cells in the pancreas of the Sunda porcupine (Hystrix javanica) and its immunolocalization. MATERIALS AND METHODS: Five adult H. javanica were used without sexual distinction. The presences of endocrine cells (glucagon, insulin, somatostatin, and pancreatic polypeptide [PP]) in pancreatic tissues were detected using the avidin-biotin-peroxidase complex method. RESULTS: The fusiform, round, and oval form endocrine cells were detected in the islets of Langerhans and exocrine parts. Most of the insulin cells were found in the central area, glucagon cells were identified in the central and peripheral areas, and somatostatin and PP cells were detected in the mantle area of the islets of Langerhans. Glucagon and somatostatin cells were also detected in smaller numbers of peripheral parts of the islet. In all of the islet parts, glucagon endocrine cells were most prevalent cell type and then, somatostatin, insulin, and PP. In the exocrine parts, PP, somatostatin, glucagon, and insulin endocrine cells were found in the inter-acinus part with moderate, moderate, a few and rare numbers, in that order. In the pancreatic duct, glucagon and somatostatin cells were found between epithelial cells in rare numbers. CONCLUSION: The pancreas of Sunda porcupine (H. javanica) contains four types of major pancreatic endocrine cells with approximately similar distribution patterns to the other rodents, except for abundant glucagon cells in the peripheral area of the islets of Langerhans.

The structural and functional recovery of pancreatic ß-cells in type 1 diabetes mellitus induced mesenchymal stem cell-conditioned medium.

AIM: Various studies have shown that secreted factors alone in culture medium without stem cell are capable of repairing tissues by itself in various conditions involving damaged tissue/organ. Therefore, this study was aimed to investigate the role of human umbilical cord mesenchymal stem cell-derived conditioned medium (CM) on the recovery of pancreatic ß-cells in Wistar rats (Rattus norvegicus) with type 1 diabetes mellitus. MATERIALS AND METHODS: The 0.05 ml CM induction was applied to the diabetic group of rats in weeks 1, 2, 3, and 4. 1 week after each CM induction, insulin concentration was analyzed using ELISA. The pancreas was divided into 3 regions, processed by paraffin method, stained with hematoxylin-eosin, and immunohistochemical method for insulin. RESULTS: This study indicated the decrease in the total number of islets and insulin concentration after the injection of single dose of alloxan. The exocrine acini were also damaged. Microscopic observation detected the presence of small islets in the diabetic group 1 week after the first 0.05 ml CM induction. The number and size of the islets increased in line with the CM doses and time of inductions. Immunohistochemically, the presence of low intensity of insulin-positive cells could be recognized at the splenic and duodenal regions of the pancreas, but not gastric region, 1 week after the first and second 0.05 ml CM induction. The intensity of staining and the number of insulin-positive cells increased dramatically in 1 week after the third and fourth 0.05 ml of CM induction in all regions of the pancreas. The data of insulin blood concentration showed clear differences between the second and the fourth induction of 0.05 ml CM induction. CONCLUSIONS: This study showed very strong evidence on the role of human umbilical cord mesenchymal stem cell-derived CM in recovering the pancreatic ß-cells damage in Wistar rats (R. norvegicus) with type 1 diabetes mellitus, structurally and functionally.

Structure and functions of the placenta in common minke (Balaenoptera acutorostrata), Bryde's (B. brydei) and sei (B. borealis) whales.

The structure and functions of placentas were examined in 3 species of rorqual whales, common minke (Balaenoptera acutorostrata), Bryde's (B. brydei) and sei (B. borealis) whales, with the aim of confirming the structural characteristics of the chorion, including the presence of the areolar part, and clarifying steroidogenic activities and fetomaternal interactions in the placentas of these whales. Placentas were collected from the second phase of the Japanese Whale Research Program under Special Permit in the North Pacific (JARPN II). Histological and ultrastructural examinations revealed that these whale placentas were epitheliochorial placentas with the interdigitation of chorionic villi lined by monolayer uninucleate cells (trophoblast cells) and endometrial crypts as well as folded placentation by fold-like chorionic villi. Moreover, well-developed pouch-like areolae were observed in the placentas, and active absorption was suggested in the chorionic epithelial cells of the areolar part (areolar trophoblast cells). Berlin blue staining showed the presence of ferric ions (Fe(3+)) in the uterine glandular epithelial cells and within the stroma of chorionic villi in the areolar part. An immunohistochemical examination revealed tartrate-resistant acid phosphatase (TRAP; known as uteroferrin in uteri) in the cytoplasm of glandular cells and areolar trophoblast cells. This result suggested that, in cetaceans, uteroferrin is used to supply iron to the fetus. Furthermore, immunoreactivity for P450scc and P450arom was detected in trophoblast cells, but not in areolar trophoblast cells, suggesting that trophoblast cells synthesize estrogen in whale placentas. Therefore, we herein immunohistochemically revealed the localization of aromatase and uteroferrin in cetacean placentas during pregnancy for the first time.

Areolae of the placenta in the Antarctic minke whale (Balaenoptera bonaerensis).

In this study, we examined the existence and structure of areolae and the steroidogenesis of areolar trophoblast cells in the Antarctic minke whale placenta morphologically and immunohistochemically. Placentas were collected from the 15th, 16th and 18th Japanese Whale Research Program under Special Permit in the Antarctic (JARPA) and 1st JARPA II organized by the Institute of Cetacean Research in Tokyo, Japan. The opening and cavity of fetal areolae formed by taller columnar trophoblast cells (areolar trophoblast cells) with long microvilli and a bright cytoplasm, as compared with the trophoblast cells of the chorionic villi interdigitating with the endometrial crypts, were recognized in observations of serial sections. The opening of the areolar cavity was hidden by chorionic villi with areolar trophoblast cells. Furthermore, a closed pouch-like structure lined by tall columnar cells similar to areolar trophoblast cells within the stroma of chorionic villi was noticed and continued to the areolar cavity, with the opening seen on serial sections. In a surface investigation of the chorion and endometrium by SEM, maternal (endometrial) areolae irregularly surrounded by endometrial folds were obvious. Moreover, we distinguished areolar trophoblast cells with long microvilli attached with many blebs from trophoblast cells. In our immunohistochemical observations, a steroidogenic enzyme, cytochrome P450 side chain cleavage enzyme (P450scc), was detected with strong immunoreactivity in trophoblast cells. However, areolar trophoblast cells showed weak or no immunoreactivity for P450scc.

Structure and steroidogenesis of the placenta in the Antarctic minke whale (Balaenoptera bonaerensis).

There are few reports describing the structure and function of the whale placenta with the advance of pregnancy. In this study, therefore, the placenta and nonpregnant uterus of the Antarctic minke whale were observed morphologically and immunohistochemically. Placentas and nonpregnant uteri were collected from the 15th, 16th and 18th Japanese Whale Research Programme with Special Permit in the Antarctic (JARPA) and 1st JARPA II organized by the Institute of Cetacean Research in Tokyo, Japan. In the macro- and microscopic observations, the placenta of the Antarctic minke whale was a diffuse and epitheliochorial placenta. The chorion was interdigitated to the endometrium by primary, secondary and tertiary villi, which contained no specialized trophoblast cells such as binucleate cells, and the interdigitation became complicated with the progress of gestation. Furthermore, fetal and maternal blood vessels indented deeply into the trophoblast cells and endometrial epithelium respectively with fetal growth. The minke whale placenta showed a fold-like shape as opposed to a finger-like shape. In both nonpregnant and pregnant uteri, many uterine glands were distributed. The uterine glands in the superficial layer of the pregnant endometrium had a wide lumen and large epithelial cells as compared with those in the deep layer. On the other hand, in the nonpregnant endometrium, the uterine glands had a narrower lumen and smaller epithelial cells than in the pregnant endometrium. In immunohistochemical detection, immunoreactivity for P450scc was detected in most trophoblast cells, but not in nonpregnant uteri, suggesting that trophoblast epithelial cells synthesized and secreted the sex steroid hormones and/or their precursors to maintain the pregnancy in the Antarctic minke whale.

Localization and expression of gastrin-releasing peptide (GRP) in the bovine cervix.

Gastrin-releasing peptide (GRP) has been suggested as a novel regulatory peptide in the female reproductive tract but the presence of GRP and GRP mRNA in the non-neurogenic tissue of the cervix has not yet been clarified. In the present study, immunohistochemistry and in situ hybridization were used to reveal the distribution of GRP immunoreactivity and expression of GRP mRNA in the bovine cervix. The cervixes from 21 non-pregnant and 20 pregnant cows, and 6 fetuses were used in the study. In the fetus, adult non-pregnant and pregnant specimens, GRP and GRP mRNA were predominantly detected in the luminal epithelial cells of basal areas of peripheral regions of the cervix. Positive staining of GRP in the epithelial cells of the cervix was first detected in the CRL 37 cm of the fetus. During the estrous cycles, the staining intensity of GRP in the epithelial cells was stronger in the follicular phase than in the luteal phase. During the early gestational period, GRP immunoreactivity was detected at relatively similar intensity to the follicular phase. In situ hybridization results ascertained the expression of GRP mRNA in the superficial epithelial cells of the cervix of non-pregnant and pregnant cows. The results suggest that GRP may be important both in the development of the fetal cervix and secretory activity of the epithelial cells of the cervix.

Expression of gastrin-releasing peptide (GRP) in the bovine uterus during the estrous cycle.

Gastrin-releasing peptide (GRP) has been proposed as a novel regulatory peptide in the reproductive tract. We previously demonstrated that GRP immunoreactivities are found predominantly in the uterine gland epithelial cells of nonpregnant and pregnant cows. The present study focused on the distribution of GRP immunoreactivity and the expression of GRP mRNA in the bovine endometrium during the estrous cycle. Tissues were collected from 21 uterine horns and bodies during the estrous cycle. RT-PCR showed the expected GRP mRNA fragments (284 bp) in the tissues from all stages of the cycle. In situ hybridization results ascertained the expression of the GRP mRNA in the uterine gland epithelial cells and superficial epithelial cells of the endometrium. Positive staining of GRP immunoreactivity in the uterine gland epithelial cells was detected in both the uterine horn and body from all stages of the cycle. In metestrus and diestrus stages, GRP was also detected in the superficial epithelial cells of horn, but not in the body. The degrees of GRP mRNA expression and intensities of GRP immunoreactivity in the endometrium increased from proestrus to diestrus stages. These findings suggest that GRP may be important both in the endometrial remodeling during the estrous cycle and in the implantation and development of blastocysts.