Seasonal changes in spermatogenesis and testicular steroidogenesis in wild male raccoon dogs (Nyctereutes procynoides)

Testes of 15 wild adult male raccoon dogs (Nyctereutes procynoides) obtained from September 2000 to April 2001 were studied to clarify seasonal changes in spermatogenesis and testicular steroidogenesis. There were marked seasonal variations in the testis weight and size with values relatively low in September and highest in March. Spermatogonia and primary spermatocytes were observed in September, while spermatogonia, spermatocytes and round spermatids were present in January, and all types of spermatogenic cells including mature spermatozoa were found in the mating season (February and March). The number of spermatogenic cells reached their peak values in February and March. In addition, steroidogenic enzymes were immunolocalized using polyclonal antisera raised against bovine adrenal cholesterol side-chain cleavage cytochrome P450 (P450scc), human placental 3beta-hydroxysteroid dehydrogenase (3 betaHSD), porcine testicular 17alpha-hydroxylase cytochrome P450 (P450c17), and human placental aromatase cytochrome P450 (P450arom). P450scc and P450c17 were identified in Leydig cells and spermatids in February, whereas these enzymes were present only in Leydig cells in September. 3betaHSD was found in Leydig cells in September and February with more intense staining in February. The localization of P450arom changed seasonally: no immunostaining in September; more extensive immunostaining in Leydig cells, Sertoli cells, and elongating spermatids in February. These results suggest that seasonal changes in the testis weight and size of wild male raccoon dogs are correlated with changes in spermatogenesis. Seasonal changes in testicular steroidogenesis suggest that the synthesis of androgen and estrogen reaches its peak in the mating season.     

Expression of steroidogenic enzymes and synthesis of steroid hormones during development of ovarian follicles in prepubertal goats

Expression of mRNAs encoding cytochrome P450 side-chain cleavage (P450scc), cytochrome P450 17 -hydroxylase (P450c17), and cytochrome P450 aromatase (P450arom) were characterized by the RT-PCR technique and concentrations of progesterone (P4), testosterone (T0) and estradiol (E2) were measured by radioimmunoassay during follicular development of prepubertal goats. Synthesis of mRNAs encoding P450scc and P450c17 began in preantral follicles, but mRNA encoding P450arom was not detectable until early antral formation. While mRNA for P450scc was expressed in both theca and granulosa cells, mRNA for P450c17 was expressed only in theca cells while P450arom mRNA only in granulosa cells. In nonatretic follicles from prepubertal ovaries, the relative quantity of mRNA expression of all the three enzymes increased with follicle size; however, while the concentration of P4 and E2 increased, that of T0 decreased with follicle size. While expression of mRNA encoding P450scc was unaffected, that of P450c17 mRNA decreased to the lowest level and mRNA for P450arom became undetectable following atresia; accordingly, while the concentration of P4 increased in the atretic medium follicles, that of T0 and E2 decreased to the lowest level after atresia. While the adult follicular stage follicles showed a similar cytochrome expression as the nonatretic follicles of prepubertal goats, the former contained higher levels of E2 and P4 than the latter. The presence of corpus luteum in an ovary decreased expression of P450scc, significantly in large follicles while it increased concentration of P4. These findings indicated that (1) similar to other species, changes in follicular steroid production in goats were explained in large measure by changes in steroidogenic enzyme expression; (2) while mRNA expression was similar, activities of some of the steroidogenic enzymes may differ between sexually mature and immature goats.     

Immunolocalization of steroidogenic enzymes in the corpus luteum and placenta of the Japanese Shiba goat

In this study, we performed immunohistochemistry of cholesterol side-chain cleavage cytochrome P450 (P450scc), 3beta-hydroxysteroid dehydrogenase (3betaHSD), cytochrome 17alpha-hydroxylase P450 (P450c17), and cytochrome P450 aromatase (P450arom) in the corpus luteum and placenta of Shiba goats. The aim was to clarify the steroidogenic capability of the corpus luteum and placenta of Shiba goats. Ovaries containing corpora lutea were obtained from four adult Shiba goats during the luteal phase (day10; n=2) and pregnancy (90 and 120 days of gestation). Placenta was obtained from one Shiba goat on day 120 of gestation. The sections of the ovaries and placentae were immunostained using the avidin-biotin-peroxidase complex method (ABC) with polyclonal antibodies generated against steroidogenic enzymes of mammalian origin. All luteal cells expressed P450scc, 3betaHSD, P450c17 and P450arom. The distribution of P450scc, 3betaHSD, P450c17 and P450arom were not different during the luteal phase and pregnancy. P450arom showed a weak positive staining in late pregnancy (120 days). In addition, immunoreactions for P450c17 and P450arom were observed in syncytiotrophoblast of the placenta of one Shiba goat. These results indicate that, in Shiba goats, corpus luteum is not only an important source of progesterone but also has the ability to synthesize androgen and estrogen during the luteal phase and pregnancy. Also the placenta has the ability to synthesize androgen and estrogen in late pregnancy.     

Distribution of Cytochrome P450-side Chain Cleavage in the Theca Interna Layers of Bovine Small Antral and Cystic Follicles

Cystic follicle is anovulatory follicular structure that is caused by an endocrine imbalance. The activity of cytochrome P450‐side chain cleavage (P450scc) is essential for the initiation of steroidogenesis in the follicle. The present study was designed to compare the frequency of cells containing P450scc between healthy and atretic small antral follicles, and among several types (I, II and III, classified based on the presence of granulosa layer) of cystic follicles. Paraffin sections of healthy (2–5 mm in diameter), atretic (2–5 mm) and cystic follicles (>25 mm) were immunohistochemically stained with rabbit polyclonal antibody to bovine P450scc. The P450scc‐positive cells were counted in four different regions of the follicles from the apical to the basal side. In small antral follicles and cystic follicles, P450scc‐positive cells were localized in the theca interna layers but not granulosa layers. The P450scc‐positive cell populations decreased in the late atretic follicles compared with the early and advanced atretic follicles at all the regions of follicle. Type III cystic follicles showed significantly lower frequencies of P450scc‐positive cells than those in the types I and II cystic follicles. These results suggest that in both small and cystic follicles in cows, total loss of granulosa cells may be associated with the reduction of frequency of P450scc‐positive cells in theca interna layer.     

Immunolocalization of steroidogenic cells in small follicles of the chicken ovary: Anatomical arrangement and location of steroidogenic cells change during follicular development

Previous anatomical and histochemical studies suggested that interstitial cells were the only steroidogenic cells in the theca layer of small follicles of the chicken ovary. However, the precise cellular site of steroid production in the small follicles is not certain. Therefore, our goal was to identify steroidogenic cells in small follicles (< 10 mm in diameter) of the chicken ovary which have not entered the follicular hierarchy by localizing steroidogenic enzymes with immunocytochemistry. Polyclonal antisera used were anti-cholesterol side-chain-cleavage cytochrome P450 (P450scc), anti-17-hydroxylase cytochrome P450 (P450c17), and anti-aromatase cytochrome P450 (P450arom) for pregnenolone-, androgen-, and estrogen-producing cells, respectively. Ovaries were collected 2 hr after oviposition and embedded in Paraplast after fixation with 4% paraformaldehyde, 10% formaldehyde, or Bouin's solution. Tissues were sectioned (4–6 μm) and sections were mounted on poly-L-lysine coated slides. Sections were incubated overnight at room temperature with each specific antiserum raised in rabbits against cytochrome P450 steroidogenic enzymes or normal rabbit serum as a control and were immunostained with an avidin-biotin-peroxidase complex. Immunoreactivity for the P450 enzymes was absent in the granulosa layer but was present in the theca layer of the small follicles (< 10 mm in diameter). Interstitial cells in the single theca layer of cortical follicles embedded in the ovarian cortex (less than 1 mm in diameter) contained P450scc and P450c17. Cells which contained P450arom, identified as aromatase cells, surrounded the interstitial cells in the theca layer. In small white follicles (approximately 1 mm in diameter), large white follicles (approximately 2–4 mm in diameter), and small yellow follicles (approximately 5–10 mm in diameter) which protruded from the surface of the ovary, the theca layer is divided into the theca interna and the theca externa. P450scc and P450c17 were localized in interstitial cells in the theca interna and externa whereas P450arom was localized in aromatase cells of the theca externa. With follicular development, more interstitial cells staining for P450scc and P450c17 appeared in the theca interna than in the theca externa whereas aromatase cells staining for P450arom were localized only in the theca externa. The distance between interstitial cells and aromatase cells within the theca layer increased as the follicles matured, resulting in a change in the anatomical relationship of steroidogenic cells. Our results of immunolocalization of cytochrome P450 steroidogenic enzymes in developing small follicles suggest that: 1) granulosa cells in small follicles are steroidogenically inactive; 2) steroids are produced in two distinct cell populations in the theca layer of small follicles, namely interstitial cells and aromatase cells; and 3) the anatomical relationship and location of interstitial cells and aromatase cells in the theca layer change with follicular maturation (a two-cell model for steroidogenesis in small follicles during follicular development).     

Immunohistochemical localization of steroidogenic enzymes in the testis of Hokkaido Sika deer (Cervus nippon yesoensis)

The testes from 15 adult male Hokkaido Sika deer (Cervus nippon yesoensis) were collected during the rutting season (October and November). We investigated the localization of 4 kinds of steroidogenic enzymes (P450scc, 3betaHSD, P450c17 and P450arom) immunohistochemically in these testicular samples. The specific immunoreactivities to these enzymes were detected only in the cytoplasm of Leydig cells. This differs to the enzyme distributions reported previously in Japanese black bear, Japanese raccoon dog, Hokkaido brown bear and American black bear, in which the same immunoreactivities were detected in Leydig cells, Sertoli cells and/or spermatogenic cells. The current study suggests that in the testes of the Hokkaido Sika deer, testosterone and estradiol-17beta may be synthesized in the Leydig cells only.     

Immunolocalization of steroidogenic enzymes in equine fetal adrenal glands during mid-late gestation

To elucidate the relationship between steroidogenic hormones and developing adrenal glands, we investigated the immunolocalization of steroidogenic enzymes in equine fetal adrenal glands during mid-late gestation. Fetal adrenal glands were obtained from three horses at 217, 225 and 235 days of gestation. Steroidogenic enzymes were immunolocalized using polyclonal antisera raised against bovine adrenal cholesterol side-chain cleavage cytochrome P450 (P450scc), human placental 3beta-hydroxysteroid dehydrogenase (3betaHSD), porcine testicular 17alpha-hydroxylase cytochrome P450 (P450c17) and human placental aromatase cytochrome P450 (P450arom). Histologically, cortex and medulla cells were clearly observed in the three fetal adrenal gland tissue samples. P450scc and P450c17 were identified in cortex cells close to medulla cells and in some medulla cells in the fetal adrenal glands. P450arom was present in both cortex and medulla cells in the fetal adrenal glands. However, 3betaHSD was not found in any of the equine fetal adrenal gland tissue samples. These results suggest that equine fetal adrenal glands have the ability to synthesize androgen and estrogen, which may play an important physiological role in the development of equine fetal adrenal glands.     

Immunohistochemical localization of steroidogenic enzymes and prolactin receptors in the corpus luteum and placenta of spotted seals (Phoca largha) during late pregnancy

To study luteal function in the late gestational period of Phocidae (seals), we analyzed the localization of steroidogenic enzymes (P450scc, 3betaHSD and P450arom) and prolactin receptors in the corpora lutea of pregnant spotted seals (Larga seal; Phoca largha) immunohistochemically. P450scc, 3betaHSD and prolactin receptors were present in all luteal cells of each corpus luteum, and most luteal cells were immunostained for P450arom. Although we analyzed only two specimens, P450scc, 3betaHSD and prolactin receptors were negatively immunostained in the placentae. P450arom was present in the syncytiotrophoblast of placentae. These findings suggest that 1) the corpus luteum of the spotted seal synthesizes pregnenolone, progesterone and estrogen during late gestational period, 2) the placenta of this species do not possess the capacity to synthesize progesterone, and 3) like other terrestrial carnivores, this species requires prolactin to maintain the corpus luteum during pregnancy. These characteristics support the recent classification of family Phocidae in the order Carnivora, and suggest a relationship between prolactin and reproductive failure during the post-implantation period in pinnipeds.     

Immunolocalization of steroidogenic enzymes P450scc, 3betaHSD, P450c17, and P450arom in Göttingen miniature pig testes

The objective of this study was to investigate immunolocalization of steroidogenic enzymes in G?ttingen miniature (GM) pig testes. Testes of 6 adult GM pigs were obtained in September 1996 (n=2), February (n=2) and June (n=2), 1997. Steroidogenic enzymes were immunolocalized using polyclonal antisera raised against bovine adrenal cholesterol side-chain cleavage cytochrome P450 (P450scc), human placental 3beta-hydroxysteroid dehydrogenase (3betaHSD), porcine testicular 17alpha-hydroxylase cytochrome P450 (P450c17), and human placental aromatase cytochrome P450 (P450arom). Histologically, all types of spermatogenic cells including mature-phase spermatozoa in seminiferous tubules were observed in all testes throughout the year. Moreover, P450scc, 3betaHSD, P450c17and P450arom were identified in Leydig cells but not in Sertoli cells of all testes. These results suggested that adult GM pig testes have the ability to produce germ cells throughout the year, and the synthesis of progestin, androgen and estrogen occurs in the Leydig cells of GM pig testes.     

Immunolocalization of steroidogenic enzymes and their expression during the breeding season in the testes of wild raccoon dogs (Nyctereutes procyonoides)

The objective of this study was to investigate immunolocalization of steroidogenic enzymes 3βHSD, P450c17 and P450arom and their expression during the breeding season in wild male raccoon dogs. The testicular weight, size and seminiferous tubule diameters were measured, and histological and immunohistochemical observations of testes were performed. The messenger RNA expression (mRNA) of 3βHSD, P450c17 and P450arom was measured in the testes during the breeding season. 3βHSD was found in Leydig cells during the breeding and non‐breeding seasons with more intense staining in the breeding season. P450c17 was identified in Leydig cells and spermatids in the breeding season, whereas it was present only in Leydig cells in the non‐breeding season. The localization of P450arom changed seasonally: no immunostaining in the non‐breeding season; more extensive immunostaining in Leydig cells, Sertoli cells and elongating spermatids in the breeding season. In addition, 3βHSD, P450c17 and P450arom mRNA were also expressed in the testes during the breeding season. These results suggested that seasonal changes in testicular weight, size and seminiferous tubule diameter in the wild raccoon dog were correlated with spermatogenesis and immunoreactivity of steroidogenic enzymes and that steroidogenic enzymes may play an important role in the spermatogenesis and testicular recrudescence and regression process.     

Spermatogenesis, serum testosterone levels and immunolocalization of steroidogenic enzymes in the wild male Japanese black bear (Ursus thibetanus japonicus)

Twenty-one wild male Japanese black bears (Ursus thibetanus japonicus) were captured in the summer-autumn of 1998-2000 in the vicinity of Neo Village, Gifu Prefecture. Testes were measured, and testicular samples were biopsied and observed histologically. Four steroidogenic enzymes, i.e., cholesterol side-chain cleavage cytochrome P450 (P450scc), 3beta-hydroxysteroid dehydrogenase (3betaHSD), 17-alpha hydroxylase cytochrome P450 (P450c17), and aromatase cytochrome P450 (P450arom) were immunolocalized. Serum testosterone concentrations were measured by radioimmunoassay. Testis size changed little from 1-3 years of age, increased rapidly at 4 years, and attained its peak at 5 years. Serum testosterone concentrations ranged from 0.05 to 1.78 ng/m l, and the mean +/- standard deviation was 0.43 +/- 0.48 ng/ml. Age of sexual maturation in wild male Japanese black bears was estimated to be 3-4 years. Seasonal changes in spermatogenesis were obvious; active in June, July and August, degenerated by September. Leydig cells, Sertoli cells and germ cells have the capability of synthesizing androgen, and Leydig cells, Sertoli cells, spermatids and spermatogonia have the capability of synthesizing estrogen in Japanese black bears.     

Changes in the Immunolocalization of Steroidogenic Enzymes and the Androgen Receptor in Raccoon (Procyon lotor) Testes in Association with the Seasons and Spermatogenesis

The raccoon is a seasonal breeder with a mating season in the winter. In a previous study, adult male raccoons exhibited active spermatogenesis with high plasma testosterone concentrations, in the winter mating season. Maintenance of spermatogenesis generally requires high testosterone, which is produced by steroidogenic enzymes. However, even in the summer non-mating season, some males produce spermatozoa actively despite low plasma testosterone concentrations. To identify the factors that regulate testosterone production and contribute to differences in spermatogenetic activity in the summer non-mating season, morphological, histological and endocrinological changes in the testes of wild male raccoons should be known. In this study, to assess changes in the biosynthesis, metabolism and reactivity of testosterone, the localization and immunohistochemical staining intensity of four steroidogenic enzymes (P450scc, P450c17, 3βHSD, P450arom) and the androgen receptor (AR) were investigated using immunohistochemical methods. P450scc and P450c17 were detected in testicular tissue throughout the year. Seasonal changes in testosterone concentration were correlated with 3βHSD expression, suggesting that 3βHSD may be important in regulating the seasonality of testosterone production in raccoon testes. Immunostaining of P450arom and AR was detected in testicular tissues that exhibited active spermatogenesis in the summer, while staining was scarce in aspermatogenic testes. This suggests that spermatogenesis in the raccoon testis might be maintained by some mechanism that regulates P450arom expression in synthesizing estradiol and AR expression in controlling reactivity to testosterone.     

Characteristics of developing prolonged dominant follicles in cattle

In cattle, sub-luteal circulating progesterone induces an increase in the frequency of LH pulses, prolonged growth of the dominant follicle, increased peripheral estradiol and reduced fertility. The objective of this study was to examine the earliest stages of development of prolonged dominant follicles, to gain insight into the etiology of this aberrant condition. Heifers were treated with an intravaginal progesterone-releasing device (CIDR) from Day 4-8 post-estrus and PGF2alpha was injected on Day 6 and again 12h later (early prolonged dominant group). Follicular phase (CIDR: Day 4-6, with PGF2alpha) and luteal phase (CIDR: Day 4-8, without PGF2alpha) groups served as controls. As expected, peripheral progesterone in heifers of the early prolonged dominant group was intermediate between luteal and follicular phase groups after luteal regression (P<0.05). On Day 7, the frequency of LH pulses was higher in heifers of the follicular phase and early prolonged dominant groups than the luteal phase group (P<0.05). Dominant follicles (n = 4 per group) were collected by ovariectomy on Day 8 and were similar in size among groups (P>0.05). Estradiol and androstenedione concentrations in the follicular fluid at ovariectomy were higher in the follicular phase and early prolonged dominant groups versus the luteal phase group (P<0.01), whereas progesterone did not differ among groups (P>0.05). Granulosa cells and theca interna isolated from dominant follicles were incubated for 3h with or without gonadotropins or frozen for later analysis of mRNA for steroidogenic enzymes. Luteinizing doses (128 ng/ml) of LH and FSH increased secretion of progesterone (P<0.05) but did not affect secretion of estradiol by granulosa cells in all groups. Low (2 or 4 ng/ml) and luteinizing doses of LH increased secretion of androstenedione by theca interna to a similar extent among groups. Expression of mRNA for P450 side chain cleavage (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), P450 aromatase (aromatase) and Steroidogenic Acute Regulatory (StAR) protein by granulosa cells did not differ among groups (P>0.05). Levels of mRNA for P450scc, 3beta-HSD, 17alpha-hydroxylase (17alpha-OH) and StAR protein in theca interna were similar in the follicular phase and early prolonged dominant groups (P>0.05), but lower in the luteal phase group (P<0.05-0.1). In summary, the premature follicular luteinization observed in previous studies after prolonged periods of sub-luteal progesterone was absent in early prolonged dominant follicles, exposed to sub-luteal progesterone for 36 h, and their characteristics resembled those of control follicles during the follicular phase.     

Correlations among antral follicular echotexture,apoptosis and expression of key steroidogenic enzymes in sheep

Nineteen cycling ewes underwent transrectal ultrasonography of ovaries followed by ovariectomies during the growth phase of the first follicular wave of the interovulatory interval or the proestrus/estrus phase of the cycle. Quantitative ultrasonographic characteristics of the antrum and follicular wall in a total of forty-three ovine antral follicles were examined for correlations with the protein expression of three steroidogenic enzymes (cytochrome P450 17α-hydroxylase, CYP17; cytochrome P450 aromatase, CYP19; and 3β-hydroxysteroid dehydrogenase, 3β-HSD) determined by densitometric analysis of immunohistochemical slides, follicular dimensions, granulosa layer thickness and the percentage of apoptotic granulosa cells. Significant correlations were found between echotextural attributes of ovine antral follicles and the percentage of apoptotic granulosa cells, CYP17 expression (theca), CYP19 expression (granulosa) and 3β-HSD expression (theca cells). Computer-aided analyses of ultrasonographic images can be beneficial to the development of assisted reproductive technologies and diagnosis of hormonal imbalances without the need for ovarian biopsies or hormone assays.     

Effects of β-OH Butyrate on Bovine Granulosa and Theca Cell Function in Vitro

In this study, the effects of beta-OH butyrate (BHB) levels, associated with a negative energy balance, on bovine granulosa and theca cell function were investigated in vitro. Granulosa and theca cells of healthy large follicles (>8 mm), obtained from slaughterhouse ovaries, were cultured in serum free medium containing 0, 0.5, 1 or 1.5 mm BHB and 3 mm glucose, to mimic the situation in the early postpartum dairy cow. Hormone concentrations (progesterone, oestradiol-17beta and/or androstenedione) in spent medium and cell numbers were measured after 48 h of culture. No effects of BHB on theca cell numbers or on steroid production were observed. In granulosa cells, all BHB treatments evenly increased cell numbers (p < 0.05), while they reduced progesterone and oestradiol-17beta production per cell (p < 0.05). These effects may be attributed to the use of BHB as energy source which is however differently metabolized than glucose. Conclusively, in the presence of physiological glucose concentrations BHB can modulate granulosa but not theca cell function in vitro.     

Effects of vinclozolin administration on sperm production and testosterone biosynthetic pathway in adult male rat

The effect of vinclozolin (VCZ), used as a fungicide and known to have anti-androgenic effects on spermatogenesis and gene expression in the male rat testis was investigated. In Experiment 1, VCZ (100 mg/kg/day) or flutamide (FM, 25 mg/kg/day) was orally administered to male Holzman rats for six days. 8 days after the last administration (D8), a drastic increase in intratesticular testosterone was detected in FM (4.2-fold over control) but not in VCZ treated animals, whereas on D36 post-administration, both groups showed similar levels. Significant decreases in daily sperm production were seen in both VCZ and FM-treated rats on D36. Semiquantitative RT-PCR analysis with testicular and pituitary mRNAs on D8 revealed that LHbeta and FSHbeta mRNAs were increased in the pituitary by VCZ, as well as by FM. Among the four testicular steroidogenic enzyme genes, cytochrome P450 side chain cleavage (P450scc) and cytochrome P450 17alpha/C(17-20) lyase (P450c17) mRNAs were significantly increased, whereas 17beta-hydroxysteroid dehydrogenase type III (17betaHSD) mRNA was not changed. A significant increase in 3beta-hydroxysteroid dehydrogenase type I (3betaHSD) and a decrease in androgen receptor (AR) mRNA were observed only in FM treated rats. Immunohistochemistry demonstrated intense staining of P450scc in the interstitial cells of VCZ-treated testis on D8. In Experiment 2, hormone levels were measured at 1, 3, 6, 12 and 24 hours after VCZ (100 mg/kg) administration to Sprague-Dawley rats. Serum LH level remained constant for the first 3 hours and started to increase at 6 hrs. In contrast, serum and intratesticular testosterone levels increased 2-fold at 1 hr and maintained the level until 24 hrs. P450c17 mRNA level was 2-fold increased at all periods, whereas no obvious changes were detected in the other steroidogenic enzyme genes. Although not statistically significant, AR mRNA level increased 2-fold, 3 hrs after VCZ administration. These results indicate that VCZ affects the pituitary in a similar manner as FM, but functions differently on testicular gene expression.     

Neurosteroidogenesis in oligodendrocytes and Purkinje neurones of cerebellar cortex of dogs

The cerebellum is a steroidogenic organ that expresses steroidogenic enzymes and produces neurosteroids. Purkinje neurones appear to be the most active steroidogenic cells in the cerebellar cortex. These neurones express 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), P450 side-chain cleavage (P450scc), 17 alpha-hydroxylase/c17, 20lyase (P450c17), P450 aromatase (P450arom) and produce pregnenolone, progesterone, dehydroepiandrosterone, androstenedion, oestradion and oestrone. Oligodendrocytes are predominantly the producer of myeline protein. The oligodendrocytes were identified by immunohistochemistry using a monoclonal antibody against myeline 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), a myeline specific enzyme. In this study we have examined the distribution of 3 beta-HSD and CNPase by immunohistochemistry using monoclonal antibody in canine cerebellar cortex. The localization of oligodendrocytes within the cerebellar cortex was determined to be close to Purkinje neurones. This result suggests that endogenous progesterone synthesized de novo in the Purkinje neurone can promote myeline protein synthesis in oligodentrocytes.     

Deficient proliferation and apoptosis in the granulosa and theca interna cells of the bovine cystic follicle

The aim of the present study was to examine the frequencies of cell proliferation and death of granulosa and theca interna layers during development of cystic follicles in order to understand the mechanisms of cystic follicle formation. Paraffin sections of cystic follicles were immunostained with antibodies against proliferating cell nuclear antigen (PCNA) and cleaved caspase-3 in order to observe proliferating and apoptotic cells, respectively. The concentrations of estradiol-17beta and progesterone in the follicular fluid of these follicles were measured by ELISA. The granulosa and theca interna layers contained both PCNA- and caspase-3-positive cells, although their numbers were limited. There was significant negative correlation between the estradiol-17beta and progesterone concentrations in the follicular fluid. Regression analysis revealed no significant correlation, except for that between the PCNA-positive cells in the theca interna and the caspase-3-positive cells in the granulosa layer. These results indicate that the granulosa and theca interna cells of the cystic follicle show weak proliferative activity and low apoptotic frequency; this implies that the cystic follicle grows slowly and then maintains a static condition without degeneration, which leads to long-term persistence of the follicle.     

Expression of messenger ribonucleic acid encoding for steroidogenic acute regulatory protein and enzymes, and luteinizing hormone receptor during the spring transitional season in equine follicles

The period of spring transition, from the anovulatory to the ovulatory season, is characterized in many mares by cyclical growth and regression of large dominant follicles. These follicles produce only low concentrations of estradiol and it is thought that acquisition of steroidogenic competence by large follicles during spring transition is prerequisite in stimulating LH prior to first ovulation. In situ hybridization was used to localize and quantify expression of factors that play a key role in follicular steroidogenesis: StAR, P450scc (CYP11A1), P450c17 (CYP17), P450arom (CYP19), and LH receptor (LHr). One ovary was obtained from mares on the day after detection of an actively growing 30 mm transitional anovulatory follicle (defined as the transitional follicle), and the remaining ovary was removed at the third estrus of the breeding season on the day after the preovulatory follicle reached 30 mm in diameter (defined as the preovulatory follicle). Messenger RNAs encoding StAR, CYP11A1, and CYP17 were detected only in theca cells and CYP19 mRNA was confined to the granulosa layer. There was significantly lower expression of mRNAs for the steroidogenic enzymes, StAR (P<0.001) and LHr (P<0.05) in transitional follicles than in preovulatory follicles. In conclusion, large equine follicles during spring transition have low levels of mRNA encoding steroidogenic enzymes, StAR and LHr which will contribute to the steroidogenic incompetence of dominant follicles during spring transition and their subsequent regression.