Pregnant Mare Serum Gonadotropin Treatment Research Articles

The hedgehog-patched signaling pathway and function in the mammalian ovary: a novel role for hedgehog proteins in stimulating proliferation and steroidogenesis of theca cells

The expression of hedgehog (Hh) genes, their receptor, and the co-receptor in mice, rat, and bovine ovaries were investigated. RT-PCR of ovarian transcripts in mice showed amplification of transcripts for Indian (Ihh) and desert (Dhh) Hh, patched 1 (Ptch1), and smoothened (Smo) genes. Semi-quantitative RT-PCR and northern blot analyses showed that whole ovarian Ihh and Dhh transcripts decreased 4-24 h after hCG versus 0-48 h after pregnant mares serum gonadotrophin treatment in mice, whereas mouse Ptch1 and Smo transcripts were expressed throughout the gonadotropin treatments. Quantitative real-time RT-PCR (qRT-PCR) revealed that the expression of the Hh-patched signaling system with Ihh mRNA abundance in granulosa cells was greater, whereas Smo and Ptch1 mRNA abundance was less in theca cells of small versus large follicles of cattle. In cultured rat and bovine theca-interstitial cells, qRT-PCR analyses revealed that the abundance of Gli1 and Ptch1 mRNAs were increased (P<0.05) with sonic hedgehog (SHH) treatment. Additional studies using cultured bovine theca cells indicated that SHH induces proliferation and androstenedione production. IGF1 decreased Ihh mRNA abundance in bovine granulosa cells. The expression and regulation of Ihh transcripts in granulosa cells and Ptch1 mRNA in theca cells suggest a potential paracrine role of this system in bovine follicular development. This study illustrates for the first time Hh activation of Gli1 transcriptional factor in theca cells and its stimulation of theca cell proliferation and androgen biosynthesis.

Regulation of adiponectin and its receptors in rat ovary by human chorionic gonadotrophin treatment and potential involvement of adiponectin in granulosa cell steroidogenesis

In mammals, adiponectin and its receptors (AdipoR1 and AdipoR2) mRNAs are expressed in various tissues. However, the cellular expression and the role of adiponectin system have never been investigated in rat ovary. Here, we report the presence of adiponectin, AdipoR1 and AdipoR2 in rat ovaries, and we have investigated its role in granulosa cells. Using RT-PCR and western blot, we show that the mRNAs and proteins for adiponectin, AdipoR1 and AdipoR2 are found in the ovaries. Immunohistochemistry localized adiponectin, AdipoR1 and AdipoR2 in theca-interstitial T-I cells, corpus luteum, oocyte and less abundantly in granulosa cells. In the KGN human granulosa cell line, adiponectin mRNA and protein were undetectable; AdipoR2 was weakly expressed, whereas AdipoR1 was clearly present. Human chorionic gonadotrophin (hCG) injection (48 h) after pregnant mare serum gonadotrophin (PMSG) injection (24 h) in immature rats increased the level of adiponectin (protein) by about threefold (P < 0.05) and those of AdipoR1 by threefold (mRNA, P < 0.05) and 1.5-fold (protein, P < 0.05) in ovary, whereas the mRNA and protein levels of AdipoR2 were unchanged. Interestingly, hCG injection (48 h) after the PMSG treatment (24 h) decreased plasma adiponectin levels and increased insulin plasma levels. In vitro in primary rat granulosa cells, human adiponectin recombinant (5 microg/ml) in the presence or absence of follicle-stimulating hormone (10(-8) M, 48 h) had no effect on the steroidogenesis. However, it increased progesterone secretion (P < 0.05) by about twofold and oestradiol production (P < 0.05) by about 1.6-fold in response to insulin-like growth factor-I (IGF-I) (10(-8) M). Furthermore, it improved IGF-I-induced IGF-I receptor-beta subunit tyrosine phosphorylation and ERK1/2 phosphorylation. In basal state, human adiponectin recombinant also increased rapidly but transiently the ERK1/2, p38 and Akt phosphorylations, whereas it increased more lately the adenosine 5'-monophosphate-activated protein kinase (AMPK) phosphorylation. Thus, AdipoR1 and AdipoR2 are regulated by hCG treatment in rat ovary and adiponectin enhances IGF-I-induced steroidogenesis in granulosa cells.

Effects of Long and Short-Term Progestagen Treatments Combined with PMSG on Oestrus Synchronization and Fertility in Awassi Ewes during the Breeding Season

Synchronization of oestrus has been used to increase reproductive efficiency in most animals, including ewes. The aim of the present study was to compare the effect of the length of a progestagen treatment (12 d vs. 6 d) on synchronization efficiency (oestrus response, time to onset of oestrus and duration of oestrus) and fertility rate using fluorogestone acetate (FGA) progestagen sponge treatment with pregnant mare serum gonadotropin (PMSG) administration applied at different times of sponge removal. Ewes (n = 68) were divided into two groups; long term (LT, n = 33) and short term (ST, n = 35) groups treated with FGA progestagen sponges. At the end of intravaginal sponge treatment period the animals of each group were divided into the 3 subgroups in relation to time of PMSG (300 IU) treatment. PMSG treatment was applied 24 h before sponge removal, at sponge removal and 24 h after sponge removal for LT1 and ST1, LT2 and ST2, and LT3 and ST3, respectively. Each ewe was inseminated intra-cervically twice with skim cow milk-diluted semen (1000 × 106 motile cells/ml) 40 h and 60 h after sponge removal. Non-return rates (NRR-30) were monitored from 12 day after sponge removal to 30 day with the aid of teaser rams. Onsets of oestrus response and oestrus cessation were significantly different (P &lt; 0.05) between the ST and LT treatment groups. Synchronization of oestrus was tighter in LT than ST group. Except for oestrus cessation, other indicators studied were not different in the ST subgroups. In the ST subgroups the oestrus cessation of the ST1 (88.7 ± 15.4 h) was the shortest and differed from ST3 (120.0 ± 14.2 h) (P &lt; 0.05). No statistical difference was observed among all studied indicators for LT groups according to application time of PMSG (P &gt;). The NRR-30 and lambing rate of the ST and LT after timed AI were 35.7% and 31.0% and 32.1% and 28.6%, respectively (P &gt; 0.05).

Regulation of protein tyrosine phosphatase 4a1, B-cell translocation gene 2, nuclear receptor subfamily 4a1 and diacylglycerol O-acyltransferase 1 by follicle stimulating hormone in the rat ovary

Ovarian response to follicle stimulating hormone (FSH) and luteinising hormone (LH) leads to the formation of a mature follicle that is eventually ovulated. FSH and LH are essential for this process because they direct changes in somatic cells associated with folliculogenesis by regulating the expression of multiple genes. We hypothesised that genes induced by FSH in rat Sertoli cells would also show hormonal regulation during rat folliculogenesis. The objective of this study was to determine the expression patterns of diacylglycerol O-acyltransferase 1 (Dgat1), nuclear receptor subfamily 4a1 (Nr4a1), an anti-proliferative gene (Btg2) and a protein tyrosine phosphatase (Ptp4a1) in the ovaries of pregnant mare serum gonadotrophin (PMSG)-treated and human chorionic gonadotrophin (hCG)-treated rats. Expression of Dgat1, Nr4a1 and Ptp4a1 was induced in ovaries 4 h post PMSG treatment. When rats were treated with hCG, Dgat1, Nr4a1 and Ptp4a1 expression was induced by 12 h. Expression of Nr4a1 protein increases 12-24 h after induction of gene expression. Nr4a1 protein was observed in the granulosa, theca and luteal cells post PMSG and hCG treatment. These findings should increase our knowledge of mechanisms regulating folliculogenesis and luteinisation and demonstrate the diverse proteins that are important in ovarian function.

Localization and Action of Dragon (Repulsive Guidance Molecule b), a Novel Bone Morphogenetic Protein Coreceptor, throughout the Reproductive Axis

Bone morphogenetic proteins (BMPs) play important roles in reproduction including primordial germ cell formation, follicular development, spermatogenesis, and FSH secretion. Dragon, a recently identified glycosylphosphatidylinositol-anchored member of the repulsive guidance molecule family, is also a BMP coreceptor. In the present study, we determined the tissue and cellular localization of Dragon in reproductive organs using immunohistochemistry and in situ hybridization. Among reproductive organs, Dragon was expressed in testis, epididymis, ovary, uterus, and pituitary. In the testis of early postnatal mice, Dragon was found in gonocytes and spermatogonia, whereas in immature testes, Dragon was only weakly expressed in spermatogonia. Interestingly, pregnant mare serum gonadotropin treatment of immature mice robustly induced Dragon production in spermatocytes. In adult testis, Dragon was found in spermatocytes and round spermatids. In the ovary, Dragon was detected exclusively within oocytes and primarily those within secondary follicles. In the pituitary, Dragon-expressing cells overlapped FSH-expressing cells. Dragon was also expressed in a number of cell lines originating from reproductive tissues including Ishikawa, Hela, LbetaT2, MCF-7, and JEG3 cells. Immunocytochemistry and gradient sucrose ultracentrifugation studies showed Dragon was localized in lipid rafts within the plasma membrane. In reproductive cell lines, Dragon expression enhanced signaling of exogenous BMP2 or BMP4. The present studies demonstrate that Dragon expression is dynamically regulated throughout the reproductive tract and that Dragon protein modulates BMP signaling in cells from reproductive tissues. The overlap between Dragon expression and the functional BMP signaling system suggests that Dragon may play a role in mammalian reproduction.

Expression of epiregulin and amphiregulin in the rat ovary.

We have previously reported that the epidermal growth factor (EGF) family growth factor, epiregulin, is expressed in rat ovarian granulosa cells by induction with pregnant mare serum gonadotropin (PMSG). In this study, we report that amphiregulin, another member of the EGF family, was also induced in the rat ovary by gonadotropin treatment. Northern blot analysis revealed that PMSG treatment induced the expression of both epiregulin and amphiregulin mRNA after 24 h, but the expression then decreased 48 h after treatment. Further treatment with human chorionic gonadotropin (hCG) rapidly induced the expression of both epiregulin and amphiregulin genes and maximal levels were reached 4 h after hCG treatment. A marginal increase in amphiregulin mRNA levels was also observed 6 h after PMSG treatment. In situ hybridization revealed that epiregulin and amphiregulin mRNAs were localized in the granulosa cells of large antral follicles. These spatio-temporal expression patterns were similar to those of cyclo-oxygenase-2 (COX-2) and progesterone receptor (PR). In adult cycling rats, epiregulin and amphiregulin were strongly induced at 1800 and 2000 h on proestrus coinciding with the preovulatory LH surge. An in situ hybridization study also showed that epiregulin and amphiregulin mRNAs were detectable in the granulosa cells of preovulatory ovarian follicles at 2000 h on proestrus, where transcripts of COX-2 and PR were co-localized with those of epiregulin and amphiregulin. These observations suggested that the EGF family members, epiregulin and amphiregulin, may play a role in the ovulatory process of cycling rats as well as in the induction of ovulation in immature rats.

Pregnancy-associated plasma protein-a production in rat granulosa cells: stimulation by follicle-stimulating hormone and inhibition by the oocyte-derived bone morphogenetic protein-15.

Pregnancy-associated plasma protein-A (PAPP-A) is the major IGF binding protein-4 (IGFBP-4) protease in follicular fluid, consistent with its proposed role in folliculogenesis. Despite growing interest, almost nothing is known about how PAPP-A expression is regulated in any tissue. Here we show that FSH and oocytes regulate PAPP-A expression in granulosa cells (GCs). By in situ hybridization, ovary PAPP-A mRNA was markedly increased by pregnant mare serum gonadotropin treatment, and the message was localized to the membrana GCs but not cumulus GCs (CGCs) of dominant follicles. To explore the mechanism, we used primary cultures of rat GCs. Control (untreated) cells produced little or no PAPP-A spontaneously. Conversely, FSH markedly stimulated PAPP-A mRNA and protein in a dose- and time-dependent fashion. Interestingly, PAPP-A expression in isolated CGCs was also strongly induced by FSH, and the induction was inhibited by added oocytes. To investigate the nature of the inhibition, we tested the effect of oocyte-derived bone morphogenetic protein-15 (BMP-15). BMP-15 alone had no effect on basal levels of PAPP-A expression by cultures of membrana GCs or CGCs. However, BMP-15 markedly inhibited the FSH stimulation of PAPP-A production in a dose-dependent manner. The cleavage of IGFBP-4 by conditioned media from FSH-treated GCs was completely inhibited by anti-PAPP-A antibody, indicating the IGFBP-4 protease secreted by GCs is PAPP-A. These results demonstrate stimulatory and inhibitory roles for FSH and BMP-15, respectively, in regulating PAPP-A production by GCs. We propose that FSH and oocyte-derived BMP-15 form a controlling network that ensures the spatiotemporal pattern of GC PAPP-A expression in the dominant follicle.

Improving the reproductive efficiency, pregnancy diagnosis and monitoring the resumption of luteal activity in indigenous damascus goats.

Seventy-five female Damascus goats aged between 1.5 and 5.5 years were used to evaluate the effectiveness of the intravaginal sponges and prostaglandin analogue on oestrous synchronization and fecundity; to diagnose pregnancy and to monitor the resumption of the luteal activity. Females were divided randomly, during the breeding season, into three equal groups, S, P and C. Females in group S were fitted with sponges containing 45 mg of flugestone acetate (FGA) for 14 days and injected with pregnant mare serum gonadotrophin (PMSG) at the sponge withdrawal. Females in group P were given two injections of prostaglandin F2alpha analogue at 11-day intervals, whereas females in group C (control) received no treatment. The results showed that there was a significant difference (p < 0.001) in oestrous exhibition between females in group S as compared with those in groups P and C, with means being 30 +/- 10, 172 +/- 115 and 217 +/- 75 h for groups S, P and C, respectively. Kidding rates resulting from the first and all matings were 80 and 88, 52 and 88, and 68 and 80% for groups S, P and C, respectively. Fecundity rates were 215, 175 and 180% for groups S, P and C, respectively, with a significant difference (p < 0.05) between the S and both P and C groups. Using an ultrasound pregnancy detector performed on days 57 +/- 3 after mating, positive pregnancy diagnosis was 93.3% and 100% for non-pregnancy. Females in the control group showed functional corpus luteum starting in September. It is concluded that FGA sponges plus PMSG treatment could be successfully used to synchronize oestrus and improve fecundity; whereas prostaglandin treatment was not effective to synchronize oestrus. It is also concluded that pregnancy can be diagnosed accurately and successfully using an ultrasound pregnancy detector. In addition, ovarian activity in the Damascus goat in Syria resumes in September.

Administration of pregnant mare serum gonadotropin to Japanese quail (Coturnix coturnix japonica): dose response over seven days and comparison of delivery by daily injection or osmotic pump

AIMS: This study is part of a research programme that aims to develop a method of hormone treatment to stimulate breeding in female birds. The aims of this study were to compare dose rates and two different delivery methods, daily injection or osmotic pump, for hormone treatment of Japanese quail (Coturnix coturnix japonica) with pregnant mare serum gonadotropin (PMSG). METHODS: PMSG (0, 5, 10, 20, 40 and 80 IU PMSG/day) was administered to 6-week-old Japanese quail housed under short-day, cool-temperature conditions (8L:16D at 7–10°C) by daily injections or osmotic pump for 7 days. Three additional groups were untreated: one group was dissected at Day 0, and two groups were maintained under either short-day, cool-temperature or long-day, warm-temperature (16L:8D, 20°C) conditions for 7 days. Cloacal diameter was measured daily, and ovarian and oviductal mass and plasma oestradiol concentrations measured at the end of the treatment period. RESULTS: PMSG treatment stimulated ovarian and oviductal growth. After 7 days of treatment with 10–20 IU PMSG, ovarian and oviductal mass were similar to those in birds moved from short to long days. Females treated with the highest doses of PMSG (40 or 80 IU) had significantly larger cloacal diameters and ovarian and oviductal mass than other treated birds or birds maintained under long-day, warm-temperature conditions. Daily injections and osmotic pumps were equally effective methods of delivery. However, there was considerable variation in response to PMSG among individual birds and this was particularly obvious at the higher doses (20–80 IU PMSG). There were no differences in plasma oestradiol concentrations between groups treated using daily injections or osmotic pumps. CONCLUSIONS: A dose of 10 IU PMSG/day was chosen for use in future experiments with Japanese quail, for the first 7 days of treatment. The delivery method of choice for future studies will depend on the practical considerations of the research in question.

Immunocytochemical localization of atrial natriuretic factor (ANF) in rat female reproductive tract: evidence for a potential hormonal regulation

In the present studies atrial natriuretic factor (ANF) was characterized immunocytochemically in the reproductive tract of immature female rats, and changes of ANF levels in response to different hormonal conditions were demonstrated. Administration of pregnant mare serum gonadotropin (PMSG) to immature animals has shown to be a useful method to synchronize growth, differentiation and atresia of ovarian follicles. ANF immunoreactivity was investigated in rat uterus and oviduct during follicular growth and estrogenic dominance (48 h after PMSG treatment) and during follicular atresia and progesterone dominance (96 h after PMSG treatment). Our immunocytochemical results showed that in rat uterus ANF was localized in endometrial mucosal and glandular epithelium and smooth muscle cells of the myometrium. In the oviduct ANF immunoreactivity was observed in mucosal cells and muscle layers. Immunocytochemical staining patterns and Western blot analysis revealed that ANF levels in rat uterus and oviduct are modulated by the hormonal status. ANF immunoreactivity was elevated during estrogenic dominance (48 h after PMSG) in uterus and oviduct. However, during progesterone dominance (96 h after PMSG) elevation of ANF immunoreactivity was observed in the uterus only. These results raise the possibility that ANF expression in rat oviduct is positively controlled by estrogen and negatively by progesterone. ANF staining in uterus during progesterone phase provides evidence that both estrogen and progesterone regulate ANF levels in uterus. The observed staining patterns indicate that ANF may have intracellular functions as well as a role in priming the extracellular environment. Accordingly, the possibility that ANF might be an important regulatory molecule for autocrine / paracrine communication within the female reproductive tract should be considered.

In Vivo Imaging of Physiological Angiogenesis from Immature to Preovulatory Ovarian Follicles

To develop a model for the study of physiological angiogenesis, we transplanted ovarian follicles onto striated muscle tissue and analyzed the process of microvascularization in vivo using repeated fluorescence microscopy. Follicles were mechanically isolated from unstimulated as well as pregnant mare's serum gonadotropin (PMSG)- or PMSG/luteinizing hormone (LH)-stimulated Syrian golden hamster ovaries and were transplanted as free grafts into dorsal skinfold chambers of untreated or synchronized hamsters. Follicles lacking thecal cell layers did not vascularize regardless whether harvested from unstimulated or PMSG-stimulated animals, but underwent granulosa cell apoptosis, as indicated in vivo by nuclear condensation and fragmentation of bisbenzimide-stained follicular tissue. In contrast, all follicles at 48 hours after PMSG treatment with a multilayered thecal shell exhibited initial signs of angiogenesis within 3 days. Vascularization was completed within 7 to 10 days, comprising a dense glomerulum-like microvascular network. Nature and extent of vascularization of follicles harvested at 72 hours after either PMSG or PMSG/LH treatment did not notably differ from each other when transplanted into the respective synchronized animals. However, follicles with PMSG/LH treatment revealed significantly larger microvessel diameters and higher capillary blood perfusion compared to follicles with sole PMSG treatment, probably reflecting the adaptation to the increased functional demand upon the LH surge. Using the unique experimental approach of ovarian follicle transplantation in the dorsal skinfold chamber of Syrian golden hamsters, we could show in vivo the developmental stage-dependent vascularization of follicular grafts with sustained potential to meet their metabolic demand by increased blood perfusion.

Expression of Ovarian Prolactin Receptor in Relation to Hormonal Changes during Induction of Ovulation in the Rat

We examined the relationships between the expression of the short and long forms of the prolactin (PRL) receptor (PRLR) mRNA in the ovary and changes in the levels of serum hormones such as sex steroid hormones and PRL during induction of ovulation in the rat. The expression of both forms of PRLR mRNA in the ovary was examined by Northern blot analysis in immature female rats treated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). Ovarian tissues and blood samples were obtained before treatment, 24 and 48 h after PMSG injection and 4, 6, 8, 12, 24 and 48 h after hCG treatment. Serum levels of 17β-estradiol, progesterone and PRL were determined by radioimmunoassay. Serum levels of 17β-estradiol rapidly increased to a maximal level 48 h after PMSG injection and then rapidly declined until 4 h after hCG injection. Serum levels of progesterone gradually increased after PMSG treatment, markedly increased to 114.2 nmol/l 8 h after hCG treatment and remained high until 48 h after hCG treatment. The serum level of PRL peaked at 66.2 µg/l (p < 0.01) 48 h after PMSG injection, and a temporary decrease after hCG treatment was followed by a continuously high level from 8 to 48 h. The expression of the long form of PRLR mRNA increased significantly (p < 0.01) to 688% of the control level after PMSG treatment, while that of the short form increased to only 184% of the control level. The expression of the long form of PRLR-mRNA rapidly declined until 6 h and then gradually increased until 48 h after hCG treatment. On the other hand, the expression of the short form of PRLR mRNA decreased to a nadir 12 h after hCG injection and then increased significantly (p < 0.01) to 142% of the control level. Our results showed that the changes in the short and long forms of PRLR mRNA differed in a time-specific manner and that these two forms are involved in different functions in the rat ovary during induction of ovulation. It is thought that the long form of PRLR mRNA is involved in folliculogenesis, while the short form of PRLR mRNA may play an important role in the formation and maintenance of the corpus luteum in the rat ovulatory cycle.

Reproductive performance of multiparous rabbit lactating does: effect of lighting programs and PMSG use.

This study aims to determine if Pregnant Mare Serum Gonadotrophin (PMSG), used for oestrous synchronization in multiparous lactating does, could be replaced by one of the following lighting schedules without impairing reproductive performance: (a) 12-h L (light)/12-h D (dark) or (b) 8-h L/16-h D, until day 6 before artificial insemination (AI), when in both cases photoperiod was changed to 16-h L/8-h D and maintained until the day of AI, and in the following 4 days post Al the light hours were progressively reduced to the initial schedules. Two groups of 20 does each were respectively submitted to one of the lighting schedules specified above. All does were artificially inseminated in 6 consecutive cycles at 42 days intervals. In the first, third and fifth AIs, PMSG (20 IU/doe via sc 48 h before AI) was used in the two groups of does, whereas in the second, fourth and sixth Als no hormonal treatment was used. Degree of oestrous synchronization (also referred in text as sexual receptivity) was estimated by the colour of the vulva at AI. Reproductive performance of does was evaluated based on fertility (kindling rates), prolificity, mortality at birth, mortality at 21 days post birth, weight of the litter at 21 days post birth and number of weaned rabbits. Oestrous was better synchronized when PMSG was used with any of the two lighting programs. Without using PMSG, a photoperiod of 12-h L/12-h D until 6 days before AI resulted in a better sexual receptivity of does than 8-h L/16-h D. Fertility, prolificity, mortality of young rabbits at 21 days, the weight of the litters at 21 days and the number of weaned rabbits did not vary with the lighting program and were not affected by the PMSG treatment. Mortality at birth, however, was higher (+1 dead kit per litter) in litters housed under a light program of 12-h L/12-h D. Global productivity (number of weaned rabbits per 100 inseminated does) was better when using PMSG, for both lighting schedules. When using a photoperiod of 12-h L/12-h D until 6 days before AI, and omitting the PMSG treatment, global productivity was scarcely reduced, however, it was considerably impaired when using a photoperiod of 8-h L/16-h D until 6 days before AI and no PMSG treatment.

Pentobarbital and ketamine suppress serum concentrations of sex hormones in the female rat.

To investigate the potential effects of pentobarbital and ketamine on serum concentrations of sex hormones, the present study was performed using pregnant mare serum gonadotropin (PMSG)-primed cyclic female Sprague-Dawley rats. Pentobarbital sodium (37 mg.kg(-1), i. p.) or ketamine-hydrochloride (229 mg.kg(-1), i. m.) was injected 2 and 3 days after PMSG treatment. At 0, 1, 2, 3, 4, and 5 days after PMSG treatment, sera were collected by cardiac puncture. The serum concentrations of progesterone (P(4)), testosterone (T), and estradiol-17 beta (E(2)) were determined by radioimmunoassay. The serum concentrations of P(4) tended to be lower in both the pentobarbital- and the ketamine-treated groups compared with the control group. Significant differences were found on days 3 and 4 after pentobarbital and on days, 1, 4, and 5 after ketamine administration. Serum concentrations of T were also suppressed in both the pentobarbital and the ketamine-treated groups, whereas E(2) concentrations decreased only in the ketamine-treated group. Pentobarbital and ketamine decrease serum sex hormone concentrations in PMSG-primed female rats.

Changes in plasma testosterone and testicular transferrin concentration, testicular histology and semen quality after treatment of testosterone-depot plus PMSG to 3 dogs with asthenozoospermia.

Three dogs diagnosed as having asthenozoospermia were given three intramuscular injections of 50 mg testosterone(T)-depot plus 250 IU pregnant mare serum gonadotropin (PMSG) at 2-week intervals, and their plasma T and testicular transferrin (Tf) concentrations, testicular histology, and semen quality were examined during the period of hormone therapy. Plasma T concentrations temporarily increased, and there was a slight improvement in spermatogenesis. Increased Tf concentrations suggested that Sertoli cell function in all three dogs was promoted by hormone treatment. The results showed that semen quality, especially the percentages of motile sperm and abnormal sperm, were improved between 1 and 5 weeks after the start of T-depot plus PMSG treatment.

Effects of thymulin on spontaneous puberty and gonadotrophin-induced ovulation in prepubertal normal and hypothymic mice.

The effects of thymulin administration beginning on days 19 or 24 of age on spontaneous puberty and gonadotrophin-induced ovulation were analysed in female normal and hypothymic mice. In normal and hypothymic mice, the daily administration of thymulin at 24 days of age resulted in a delay in the age of vaginal opening, with an increase in serum progesterone levels. Normal mice treated with 200 ng thymulin beginning on day 19 of age and injected with pregnant mare serum gonadotrophin (PMSG) 24 h later had an increase in ovulation rate, number of ova shed and weight of the ovaries. None of the hypothymic mice treated with thymulin on day 19 and PMSG on day 20 ovulated. PMSG treatment on day 25 induced ovulation in hypothymic mice. When these animals were injected previously with 200 ng thymulin, the number of ova shed by ovulating animals was lower than in PMSG-treated animals. Administration of thymulin and sequential injection of PMSG and human chorionic gonadotrophin 54 h later resulted in an increase in ovulatory response in comparison with those receiving only PMSG. The results suggest that thymulin plays a role in the regulation of spontaneous puberty through its effects on adrenal and ovarian endocrine functions. The increase in the ovarian PMSG response-treated animals, previously given thymulin, showed that this thymic hormone participates in the regulation of gonadotrophin secretion mechanisms and seems to be dose- and age-dependent. In hypothymic mice, neuroendocrine mechanisms regulating puberty are different from those of normal mice.

Cellular retinoic acid-binding protein(II) presence in rat uterine epithelial cells correlates with their synthesis of retinoic acid.

Vitamin A (retinol) and retinoic acid are necessary for the maintenance of the female reproductive system of higher animals. Our previous work has demonstrated cell specific expression of cellular retinoic acid-binding protein (CRABP) and cellular retinoic-acid binding protein(II) [CRABP(II)] in the uterus of the rat. CRABP(II) expression was shown to be induced in the uterine surface epithelial cells by treatment of prepubertal rats with pregnant mare serum gonadotropin (PMSG). Here we report that, after PMSG treatment, collected uteri had markedly higher levels of retinoic acid than did the uteri of prepubertal rats treated with the control vehicle. Smooth muscle, stromal, and epithelial cells were then cultured from uteri from such animals and provided with retinol or with the retinol/retinol-binding protein complex. Retinoic acid production, analyzed by high-performance liquid chromatography, was observed for the epithelial cells from the uteri of prepubertal animals treated with PMSG, cells previously shown to express CRABP(II) and confirmed here to continue to express it in culture. Little or no retinoic acid was produced by cultured epithelial cells from the prepubertal uteri [shown previously to be negative for CRABP(II)] or by smooth muscle and stromal cells taken from uteri of prepubertal or PMSG-treated rats (shown previously to express CRABP). Retinoic acid production by uterine epithelial cells [and CRABP(II) expression] was also observed if the prepubertal rat was treated with estrogen before cell collection. At no time did cells expressing CRABP exhibit significant retinoic acid synthesis. Thus, this system revealed an important difference in retinoid metabolism between cells expressing CRABP and CRABP(II) and suggests CRABP(II) may participate in retinoic acid production and/or secretion.

Changes in ovarian expression of tissue-type plasminogen activator and plasminogen activator inhibitor type-1 messenger ribonucleic acids during ovulation in rat.

We investigated the time course and localization of ovarian tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor type-1 (PAI-1) expression during the ovulatory period in rat by RNase protection assay and in situ hybridization. Immature female Wistar rats were injected with 25 IU pregnant mare serum gonadotropin (PMSG), followed 50 h later by 25 IU human chorionic gonadotropin (hCG). Levels of tPA mRNA were low before hormone treatment and after PMSG treatment. After hCG treatment, tPA mRNA levels increased rapidly, the first peak at 4 h after hCG treatment and reached a maximum just prior to ovulation, 12 h later, before declining again. PAI-1 mRNA was barely detectable before hormone treatment but was transiently induced by hCG treatment, reaching peak levels after 4 h. Subsequently, PAI-1 mRNA levels decreased until early luteinization. The expression of tPA mRNA 4 h after hCG treatment occurred mainly in the follicular thecal-interstitial cells, but was barely detectable in the granulosa cells, whereas 12 h after hCG treatment it was maximal in the granulosa cells of the large follicles destined to ovulate. PAI-1 mRNA was expressed mainly in ovarian stromal tissue and in the thecal external interstitial cells encapsulating the follicles at 4 h after hCG treatment. These results suggest that the temporal regulation of tPA biosynthesis after hCG induction depends on the cell types and size classes in the various ovarian compartments. PAI-1 may be produced by the stormal tissue and the thecal external interstitial cells and is perhaps implicated in structural changes during follicular growth, ovulation and luteinization.

EEECI OF LEAD EXPOSURE ON SOME SELECTED BIOCHEMICAL AND HAEMATOLOGICAL VARIABLES WITH SPECIAL REFERENCE TO REPRODUCTIVE TOXICITY IN FEMALE RABBITS.

The present study was designed to assess the effect of different levels of lead on ovarian activity and to elucidate some biochemical and haematological variables that can be associated with these levels. Immature female New Zealand rabbits were used, divided into four groups, each of six rabbits. Group I and II received low (5.4 mg/kg b. wt.) and high (10.8 mg / kg b.wt.) doses of lead acetate orally, respectively, daily for 5 days/week over a period of 8 weeks. Groups III and IV were kept as control. All groups (except group IV) were treated with 200 1.U pregnant mare serum gonadotrophin (PMSG) I/m, followed by i.m injection of 200 I.U human chorionic gonadotrophin (HCG), 48hrs after commencement of PMSG treatment. The results indicated that injection of PMSG followed by HCG had stimulated the ovarian activity of the control virgin does and elevated the total ovarian response, Metion rate and ovulation percentage. However, administration of low and high doses of lead acetate resulted in a significant decrease in total ovarian response, ovulation rate and ovulation percentage. Lead administration significantly decreased the levels of decteasereins and total lipids. However, a significant increase in the levels of triglycerides was obtained with low and high doses of lead. Cholesterol level was significantly increased with the high dose only. Estradiol-17β and progesterone were significantly decreased following oral administration of both doses of lead acetate. However, lead acetate administration resulted in a significant plasma of bolts were significantly increased with the low and high doses of lead acetate. However, haemoglobin concentration and response with profound changes in the biochemical, haematological and hormonal profiles

Hormonal regulation of steroidogenic acute regulatory (StAR) protein messenger ribonucleic acid expression in the rat ovary

Steroidogenic acute regulatory (StAR) protein is thought to mediate the rapid increase in steroid hormone biosynthesis in response to tropic hormones by facilitating cholesterol transport to the inner mitochondrial membrane where the P450 side-chain cleavage enzyme (P450scc) is located. Since cholesterol delivery is the regulated step in steroidogenesis and is dependent onde novo protein synthesis, StAR mRNA levels were examined in response to the tropic hormones, pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). The results of this investigation revealed that major StAR mRNA transcripts of 3.4 and 1.6 kb and a less abundant transcript of 1.2 kb were detected in the adrenal, ovary, and testis. Within the ovary, StAR mRNA levels were regulated by PMSG and hCG. The two major transcripts were increased in the immature rat ovary following PMSG administration and in the ovary, 8 d after ovulation, in response to stimulation by hCG. Serum progesterone levels were increased following hCG treatment in parallel with the enhanced expression of StAR. Following PMSG treatment, ovarian StAR transcripts at 3.4 and 1.6 kb were each increased twofold. In the ovary, 8 d following ovulation, basal ovarian StAR mRNA levels were elevated up to sixfold relative to the preovulatory StAR mRNA levels. Even with the enhanced basal level of StAR mRNA within the ovary 8 d postovulation, hCG administration still resulted in a 2.5- and 7-fold increase in the 3.4 and 1.6 kb (p<0.025) transcripts, respectively, and a 58% increase in serum progesterone. In contrast to the dramatic alterations in StAR mRNA expression following hormonal stimulation, P450scc mRNA levels remained unchanged in response to hCG stimulation. The levels of serum progesterone paralleled the change in ovarian StAR mRNA in all experiments. This study provides the first evidence that StAR mRNA expression in the rat ovary is mediated by gonadotropins, further supporting its important role in the regulation of steroid hormone biosynthesis.