Ultrastructure of rat pinealocytes in vitro: Influence of gonadotropic hormones and LH-RH

Summary The influence of gonadotropic hormones on the ultrastructure of rat pinealocytes in short-term organ culture was studied. Human chorionic gonadotropin (HCG), as well as pregnant mare serum gonadotropin (PMSG), caused a marked activation of pinealocytes. An hypothesis is discussed implying the presence of a feedback mechanism between the pineal organ and the hypothalamo-hypophysial system.     

Effects of gonadotropins on follicular development, ovulation, and atresia in the mature guinea pig

The induction of follicular growth, ovulation, and atresia by heterologous gonadotropic preparations was studied late in the reproductive cycle of the adult female guinea pig. Human chorionic gonadotropin (HCG) administration (10 IU) 12 days following the first signs of opening of the vaginal membrane was found to stimulate ovulation within 24 h in all animals studied, as evidenced by recovery of ova from their oviducts as well as the presence of postovulatory follicles in their ovaries. Histologically, ovaries of animals receiving HCG exhibited atretic changes in most of the follicles smaller than 999 micrometer in diameter. Pregnant mares serum gonadotropin (PMSG, 10 IU) administered on days 9 and 10 of the cycle was not sufficient to stimulate ovulation in this species although histological changes in the follicular complement were observed. Administration of PMSG prior to the HCG appeared to have an inhibitory effect on ovulation induction. Follicles luteinizing with entrapped ova were seen in all groups receiving exogenous gonadotropin, although they were most prevalent in the animals receiving the maximum total gonadotropin doses (i.e. PMSG + HCG).     

Response of mouse ovaries in vivo and in organ culture to pregnant mare's serum gonadotrophin and human chorionic gonadotrophin

The effect of various doses of pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (HCG) on the yield of oocytes undergoing preovulatory maturation in organ culture was studied in mice. The mice received ip injections of .5, 1, 3, or 5 IU PMSG and 48 hours later, 0, 1, 2, or 4 IU HCC or their ovaries were cultured with 0, .2, .4, or .8 IU HCG/ml. 3-5 IU PMSG by 1-2 IU HCG induced maximum preovulatory response both in vivo and in culture. The proportion of large antral follicles with oocytes at Metaphase 2 was lower in culture than in the intact animal. However, the number of preantral and small Graafian follicles increased to a higher level in vivo and thus the overall total number of oocytes that progressed beyond the germinal vesicle stage was higher in organ culture.     

Effects of gonadotropins on oocyte maturation and progesterone production by porcine ovarian follicles cultured in vitro

Effects of gonadotropins on the maturation of isolated oocytes and production of progesterone by porcine ovarian follicles from gonadotropin treated gilts have been studied in vitro. The addition of gonadotropins (2 I. U./ml, PMSG, HGC or 2 mg/ml FSH) to the culture medium resulted in increasing the number (84 - 90 %) of isolated oocytes which reached metaphase II. Expansion of the whole cumulus mass was observed only in media containing PMSG, whereas FSH or HCG alone did not cause these marked changes in the cumulus cells. Denudation of the eggs prior to culture gave no significant differences in the maturation rates between oocytes cultured in media with or without gonadotropins. In vitro maturation of follicle-enclosed oocytes took place only in HCG treated animals. Removing the ovary at 15 or 60 minutes after intravenous HCG administration induced oocyte maturation only in 22% and 17% respectively. A sharp increase in the number of oocytes which resume meiosis during follicle culture was observed 4 hours after HCG injection (84 %) and all of the oocytes of the gilts ovariectomized at 8 hours after HCG injection matured during the culture period. The progesterone production of isolated follicles from control gilts (only PMSG injected) increased slowly during a 96-hour culture period (from 48 to 240 ng progesterone/follicle), whereas the secretion of progesterone was drastically increased after a 15 minute interval between HCG injection and ovariectomy (from 42 to 950 ng progesterone/follicle). Follicles removed 24 hours after HCG injection showed a further increase in steroid production (2000 ng progesterone/follicle) and consistently secreted large amounts of progesterone during the culture period.     

SUPEROVULATION AND SUPERPREGNANCY IN THE GOLDEN HAMSTER*

Pregnant mare serum gonadotropin (PMSG) treatment given in the morning or afternoon on any day of the four-day estrous cycle and human chorionic gonado tropin (HCG) given two days later successfully induced superovulation in the golden hamster. The minimum interval between PMSG and HCG necessary to obtain consistent superovulation was approximately 44 hr. The lowest ovulation rate was obtained following PMSG treatment on the afternoon of day 4 despite the fact that this time coincides with the maximum endogenous FSH level, necessary for the maturation of the next crop of follicles destined to ovulate. Thirty-eight to one-hundred percent of superovulated females in four different treatment groups became superpregnant after natural mating. Some treated females exhibited two consecutive nights of estrus with ovulation apparently occurring during the second night. Superpregnant females delivered “super” size litters, up to 27 live-born pups. The ultimate litter size appeared to be established after day 3 and prior to day 8 of superpregnancy. A one-day extension of the normal 16-day gestation period was observed in 31% of superpregnancies. Unilateral pregnancies were observed at autopsy in 44% of treated females which received the high dose of PMSG (30 IU). The progeny of superovulated females reproduced normally at maturity. The results indicate that ova from superovulated female hamsters are capable of full normal development.     

Cytochemical studies on cytoplasmic granular elements in the hamster pineal gland.

The pineal gland of adult golden hamsters (Mesocricetus auratus) was studied by various cytochemical methods at the electron microscopic level: (1) the modified chromaffin reaction specific for 5-hydroxytryptamine (5-HT), (2) argentaffin reaction, (3) zinc-iodide-osmium (ZIO) mixture reaction and (4) acid phosphatase reaction. In the pinealocytes, the dense-cored vesicles (80-160 nm in diameter) show both chromaffinity and argentaffinity, while the population of dense bodies (150-400 nm in diameter) is reactive to ammoniacal silver solution and ZIO mixture but not to the modified chromaffin reaction. After incubation for demonstration of acid phosphatase activity, reaction products are localized in some, but not all, of the dense bodies, in some of the small vesicles in the Golgi region and in one or two inner Golgi saccules. In nerve fibers in the pineal gland, small granulated vesicles are also reactive to the modified chromaffin reaction and ZIO mixture. Based upon these cytochemical results the following conclusions have been reached: (1) dense cored vesicles in the pinealocytes and small granulated vesicles in the nerve fibers of the hamster pineal gland contain 5-HT, and (2) the population of dense bodies in the pinealocytes is heterogeneous, some are lysosomes and the other are possibly the granules responsible for the secretion of pineal peptides.     

"Light" and "dark" pinealocytes of the porcine pineal gland

Both qualitative and quantitative comparative studies of "dark" and "light" pinealocytes of the porcine pineal gland have been carried out. These cells differ from each other in their electronic density of cytoplasm, shape of nucleus, the structure of membrane bound dense bodies and the number of microtubules and smooth endoplasmic reticulum. The membrane bound dense bodies--characteristic structures of pig pinealocytes as well dense core vesicles occur in both types of cells. The relative volume of the majority of the cells' organellae apart from the Golgi apparatus, also do not show any significant difference. The results obtained support a functional basis for pinealocyte differentiation in the porcine pineal gland.     

Inability of progestogen pretreatment to prevent premature luteolysis of induced corpora lutea in the anestrous bitch

Fourteen mature anestrous bitches were used to determine the effectiveness of pretreatment with an orally active progestogen to prevent premature luteolysis of induced corpora lutea (CL) in the anestrous bitch. In Group 1, seven bitches were treated orally with megestrol acetate (Ovaban((R))) at the rate of 2.2 mg/kg body weight for eight days. Three days later, the bitches were treated daily with pregnant mare's serum gonadotropin (PMSG) (44 IU/kg body weight) administered intramuscularly for nine consecutive days, and each bitch was given 500 IU human chorionic gonadotropin (HCG) on day 10, or on the first day of induced estrus if the bitches exhibited estrus while being treated with PMSG. A control group (Group 2) of seven bitches was not treated with Ovaban((R)) but was similarly given PMSG and HCG. Estrus was detected twice daily using a vasectomized male dog and verified by vaginal cytology. Blood samples were obtained on the first day of induced estrus (day 0) and every other day until day 90 post-estrus. Plasma progesterone (P(4)) concentrations were determined by a non-extraction solid phase radioimmunoassay (RIA), and data were analyzed by Student's t-test. There was no significant difference between the progesterone profiles of both groups of bitches. In addition, P(4) values were less than 1 ng/ml by day 50 post-estrus. Results of this study suggested that pretreatment with an orally active progestogen was not effective in preventing premature luteolysis of induced CL in the anestrous bitch.     

Cytochemical studies on cytoplasmic granular elements in the hamster pineal gland

Summary The pineal gland of adult golden hamsters (Mesocricetus auratus) was studied by various cytochemical methods at the electron microscopic level: (1) the modified chromaffin reaction specific for 5-hydroxytryptamine (5-HT), (2) argentaffin reaction, (3) zinc-iodide-osmium (ZIO) mixture reaction and (4) acid phosphatase reaction. In the pinealocytes, the dense-cored vesicles (80–160 nm in diameter) show both chromaffinity and argentaffinity, while the population of dense bodies (150–400 nm in diameter) is reactive to ammoniacal silver solution and ZIO mixture but not to the modified chromaffin reaction. After incubation for demonstration of acid phosphatase activity, reaction products are localized in some, but not all, of the dense bodies, in some of the small vesicles in the Golgi region and in one or two inner Golgi saccules. In nerve fibers in the pineal gland, small granulated vesicles are also reactive to the modified chromaffin reaction and ZIO mixture. Based upon these cytochemical results the following conclusions have been reached: (1) dense cored vesicles in the pinealocytes and small granulated vesicles in the nerve fibers of the hamster pineal gland contain 5-HT, and (2) the population of dense bodies in the pinealocytes is heterogenous, some are lysosomes and the others are possibly the granules responsible for the secretion of pineal peptides.Supported in part by a grant from the National Science Council, Republic of ChinaDedicated to Professor Doctor Huoyao Wei on the occasion of his 70th birthday     

Estrus, ovulation and conception following timed insemination in Romney ewes treated with progestagen and gonadotropins.

The estrus — ovulation time relationships was examined in Romney ewes treated with progestogen (intravaginal sponge) and gonadotropins (PMSG + HCG or PMSG alone) prior to (January) and during (April) the breeding season. The conception rate of ewes inseminated at predetermined times after treatment was also investigated.Ewes exhibited estrus sooner after sponge removal in April than in January (34.9 v 38.9 hrs, P < 0.001). The interval from sponge removal to ovulation was also shorter in April than in January (56.3 – 62.1 hrs, P < 0.01). There were no significant differences between treatments or season on the mean interval from estrus to ovulation. Types of gonadotropin treatment had no effect on the estrus — ovulation time relationships. There were no significant effects of season, hormone treatment or time of insemination on lambing rate.     

Effect of gonadotrophins and testosterone on the seminiferous tubules of the immature rat.

The actions of HCG and PMSG for different periods and of testosterone of the immature rat testis were studied. Short-term administration of HCG (1-3 days) induced an early meiotic and postmeiotic stimulatory effect but a decrease in spermatogonial numbers. Administration of HCG for longer periods (10 days) caused a reduction in numbers of all cell types. Treatment with HCG + PMSG reduced the amount of inhibition, while PMSG alone resulted in histological and humoral signs of stimulation of the interstitial tissue and the meiotic and postmeiotic stages; the numbers of spermatogonia were not affected. Testosterone caused stimulation of the meiotic and postmeiotic stages and a reduced number of spermatogonia. It is concluded that while PMSG directly stimulates spermatogonia, HCG acts through testosterone secretion at the meiotic and postmeiotic stages. The early inhibitory effects of HCG and testosterone on spermatogonial numbers could be ascribed to the inhibition of endogenous FSH by androgens.     

Progesterone and estrogens in the pregnant and nonpregnant dolphin, Tursiops truncatus, and the effects of induced ovulation

Baseline serum levels of progesterone and total immunoreactive estrogens were determined for intact and ovariectomized captive female Atlantic bottlenose dolphins (Tursiops truncatus), as well as newly captured wild adult females. Stimulation of ovarian follicular growth and ovulation was attempted by intramuscular injection of pregnant mare's serum gonadotropin (PMSG). High doses of PMSG were required to increase serum estrogen levels. When PMSG was followed by an injection of human chorionic gonadotropin (hCG), ovulation was presumed to have occurred as indicated by subsequent high levels of serum progesterone. From these observations, it appears that 1) females with progesterone levels greater than 3000 pg/ml over an extended period are pregnant, 2) Tursiops truncatus is capable of spontaneous ovulation in captivity without gonadotropin therapy, 3) captive female dolphins, although relatively resistant to PMSG, can be induced to ovulate using a combination of high intramuscular-injected doses of PMSG followed by hCG, and 4) spontaneous ovulation is likely to follow an induced ovulation.     

C57BL/6J小鼠超数排卵的研究

目的　确定C57BL 6J小鼠超排的最佳激素剂量和最合适的注射间隔时间 ,提高超排率。方法　 40只C57BL 6J雌鼠随机分为四组 ,分别用 5IU或 10IU的PMSG和HCG ,间隔 48h或 72h注射 ,比较排出卵母细胞的数量。结果　 5IU +5IU剂量的PMSG和HCG、间隔 48h注射组超排效果最好 ;8～ 10周龄雌鼠较 6～ 8周龄雌鼠超排效果好。结论　C57BL 6J小鼠超排的最佳激素剂量为 5IUPMSG +5IUHCG ,最合适的注射间隔时间为 48h ,处于繁殖期的雌鼠超排效果好。     

Effects of gonadotrophin on the ovary of Apodemus flavicollis in winter anoestrus

Wild-caught female Apodemus flavicollis were given daily subcutaneous injections of PMSG + HCG on two consecutive days (2xPMSG + HCG), HCG on two days (2 X HCG), PMSG + HCG until their vagina became perforate (Perforate), and PMSG + HCG until they became perforate and were left undisturbed for another five days (Perforate + 5). Untreated females served as controls. The ovaries and uteri increased in weight as a result of the treatment. The number of healthy follicles increased in "perforate" females and decreased in "2 X HCG". Luteinization of the follicles and possibly of the interstitial tissue was seen in "2 X HCG". In this group, the mean diameter of the corpora lutea increased, and it decreased in "perforate" and "perforate + 5". The mean number of corpora lutea was unaffected by the treatment. The activity of 3 beta hydroxysteroid dehydrogenase (3 beta HSD) was strong in the corpora lutea of the untreated females and remained so throughout treatment. The interstitial tissue of the untreated females showed only weak 3 beta HSD activity, but the activity was strong in all the experimental groups. One female ovulated, but it is not clear if it was in response to treatment.     

Gonadotropin regulation of glutamate cysteine ligase catalytic and modifier subunit expression in rat ovary is subunit and follicle stage specific

We have observed that levels of the antioxidant glutathione (GSH) and protein levels of the catalytic and modifier subunits of the rate-limiting enzyme in GSH synthesis, GCLc and GCLm, increase in immature rat ovaries after treatment with gonadotropin. The goals of the present studies were to delineate the time course and intraovarian localization of changes in GSH and GCL after pregnant mare's serum gonadotropin (PMSG) and after an ovulatory gonadotropin stimulus. Twenty-four hours after PMSG, there was a shift from predominantly granulosa cell expression of gclm mRNA, and to a lesser extent gclc, to predominantly theca cell expression. GCLc immunostaining increased in granulosa and theca cells and in interstitial cells. Next, prepubertal female rats were primed with PMSG, followed 48 h later by 10 IU of hCG. GCLm protein and mRNA levels increased dramatically from 0 to 4 h after hCG and then declined rapidly. There was minimal change in GCLc. The increase in gclm mRNA expression was localized mainly to granulosa and theca cells of preovulatory follicles. To verify that GCL responds similarly to an endogenous preovulatory gonadotropin surge, we quantified ovarian GCL mRNA levels during the periovulatory period in adult rats. gclm mRNA levels increased after the gonadotropin surge on proestrus and then declined rapidly. Finally, we assessed the effects of gonadotropin on ovarian GCL enzymatic activity. GCL enzymatic activity increased significantly at 48 h after PMSG injection and did not increase further after hCG. These results demonstrate that gonadotropins regulate follicular GCL expression in a follicle stage-dependent manner and in a GCL subunit-dependent manner.     

Induction of estrus in pubertal miniature gilts

Genetic engineering of miniature pigs has facilitated the development of numerous biomedical applications, such as xenotransplantation and animal models for human diseases. Manipulation of the estrus is one of the essential techniques for the generation of transgenic offspring. The purpose of the present study was to establish a useful method for induction of the estrus in miniature gilts. A total of 38 pubertal miniature gilts derived from 4 different strains were treated with exogenous gonadotropins. Estrus and ovulatory response were examined after treatment with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) as 200 IU PMSG and 100 IU hCG, 300 IU PMSG and 150 IU hCG, or 1,500 IU PMSG only, followed by 100, 150 or 750 IU hCG 72 h later, respectively. The optimal protocol was determined to be the combination treatment of 200 IU PMSG and 100 IU hCG followed by 100 IU hCG. The administration of 200 IU PMSG and 100 IU hCG was effective in inducing estrus regardless of the strain, although there was a strain difference in the ovulatory response. These results indicate that treatment with a low-dose combination of PMSG and hCG provides one of the simplest methods for induction of estrus and ovulation in pubertal miniature pigs.     

Sequential changes associated with the degeneration of preovulatory rat follicles.

In 26-day-old rats, follicles capable of ovulation were present 48 h after PMSG injection and they degenerated if not exposed to an ovulating dose of HCG. In such follicles 125I-labelled LH bound to the thecal and granulosa cells. By 60 h after PMSG, LH binding to the granulosa cells was reduced by 46% although these follicles retained their ability to ovulate. LH binding to the granulosa cells was lost in most follicles by 72 h and ovulation could not be induced. The thecal cells still possessed LH binding sites at 72 h after PMSG. HCG stimulation of these follicles resulted in disruption of the granulosa and the invasion of blood cells into the antrum.     

Plasma levels of oestradiol-17 beta in the pre-puberal heifer treated to induce superovulation

The plasma concentrations of oestradiol-17β have been measured by radioimmunoassay in pre-puberal calves following treatments used to induce superovulation (PMSG/HCG and PMSG/FGA/HCG). Before treatment, in almost all animals, the concentrations of oestradiol-17β were different from zero (2 to 8 pg/ml). The highest concentrations were measured around 130 h. after the beginning of treatment, before ovulation (150 to 2050 pg/ml). The curves showing the changes in hormonal levels have the same form as those of follicular growth measured using morphological criteria. The two hormonal treatments resulted in similar oestradiol-17β concentrations.     

周龄和激素水平对长爪沙鼠超数排卵效果的影响

目的探讨不同周龄和激素水平对长爪沙鼠超数排卵效果的影响,以期确定长爪沙鼠最佳超排周龄和激素使用剂量。方法腹腔注射10 IU PMSG/HCG对4～18周龄8个年龄段的雌性长爪沙鼠进行超数排卵,末次注射16～17 h内对各组动物卵母细胞计数,确定最佳超排周龄后,对该年龄动物以5、10、15 IU3个剂量水平腹腔注射PMSG/HCG,观察各组动物的卵母细胞计数差异。结果与其它周龄组相比,6周龄组长爪沙鼠超数排卵后的卵母细胞数最多,各组间有统计学意义（P〈0.05）,而5、10、15IU等3个剂量组的超排效果也有一定的差异,10 IU组数量最高。结论对长爪沙鼠而言,采用10 IU激素注射和6周龄的动物进行超数排卵,获得的卵母细胞数量最多而且超排效果稳定性。     

Studies of hCG binding and endocytosis in rat ovary by ultrastructural immunocytochemistry

Summary Localization of hCG binding sites and the process of endocytosis in pseudopregnant rat ovaries were investigated by indirect electron-microscopic immunocytochemistry. Immature female rats were treated with pregnant-mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) to induce ovarian luteinization. Eight days after priming with PMSG-hCG and 1–6 h before sacrifice the animals were given another injection of hCG to bind the receptors. Receptor sites to hCG localized by reaction product were present in most luteal cells, but not in primary follicular cells. The receptor sites were distributed on luteal cell surfaces facing interstitial spaces. Endocytotic pits containing hCG binding sites were rarely seen 1 h after hCG injection. At 2 h, hCG and presumably its receptor were taken up within endocytotic vesicles with the evidence of reaction product coated on the vesicle wall. With time, fusion of endocytotic vesicles with lysosome occurred and the reaction product appeared in phagolysosomes. The reaction product was localized on phagolysosomal inner surface or in free granular form. These findings suggest that hCG and its receptors were internalized through endocytotic pits and endocytotic vesicles and delivered to lysosomes probably for degradation. An additional experiment for localization of acid phosphatase was also performed to delineate the lysosomes and phagolysosomes.