Studies on follicular growth in the immature rat and hamster: Effect of a single injection of gonadotropin or estrogen on the rate of3H-thymidine incorporation into ovarian DNAin vitro

Initiation of follicular growth by specific hormonal stimuli in ovaries of immature rats and hamsters was studied by determining the rate of incorporation of³H-thymidine into ovarian DNAin vitro. Incorporation was considered as an index of DNA synthesis and cell multiplication. A single injection of pregnant mare serum gonadotropin could thus maximally stimulate by 18 hr³H-thymidine incorporation into DNA of the ovary of immature hamsters. Neutralization of pregnant mare serum gonadotropin by an antiserum to ovine follicle stimulating hormone only during the initial 8–10 hr and not later could inhibit the increase in³H-thymidine incorporationin vitro observed at 18 hr, suggesting that the continued presence of gonadotropin stimulus was not necessary for this response. The other indices of follicular growth monitored such as ovarian weight, serum estradiol and uterine weight showed discernible increase at periods only after the above initial event. A single injection of estrogen (diethyl stilbesterol or estradiol-l7β) could similarly cause 18 hr later, a stimulation in the rate of incorporation of³H-thymidine into DNAin vitro in ovaries of immature rats. The presence of endogenous gonadotropins, however, was obligatory for observing this response to estrogen. Evidence in support of the above was two-fold: (i) administration of antiserum to follicle stimulating hormone or luteinizing hormone along with estrogen completely inhibited the increase in³H-thymidine incorporation into ovarian DNAin vitro; (ii) a radioimmunological measurement revealed following estrogen treatment, the presence of a higher concentration of endogenous follicle stimulating hormone in the ovary. Finally, administration of varying doses of ovine follicle stimulating hormone along with a constant dose of estrogen to immature rats produced a dose-dependent increment in the incorporation of³H-thymidine into ovarian DNAin vitro. These observations suggested the potentiality of this system for developing a sensitive bioassay for follicle stimulating hormone.     

Equine oocyte in vitro maturation: influences of sera, time, and hormones.

Objectives of the present research were to determine the influences of types of media, sera, time and hormones on equine oocyte in vitro maturation (IVM). The following types of media and sera were evaluated: Menezo's B2 medium (B2), modified Tissue Culture Medium 199 (TCM), Defined Medium (DM), fetal calf serum (FCS), mare serum collected on the first day of estrus (MS), and mare serum collected on the day of ovulation (MSO). Resultant oocyte maturation was compared with the control: DM with bovine serum albumin (BSA). Effect of culture time (0, 15, and 32 hr) and the following hormones on oocyte IVM were evaluated: none, bovine luteinizing hormone (bLH; 1, 10, 100 micrograms/ml), equine luteinizing hormone (eLH; 100 micrograms/ml), bovine follicle-stimulating hormone (FSH; 5 micrograms/ml), and equine chorionic gonadotropin (eCG; 1 and 100 IU/ml). Cumulus expansion in the media and sera experiments was 50% (DM with BSA), 80% (TCM, B2, and DM with MS or MSO), and 100% (FCS with any medium). The proportion of metaphase II (MII) oocytes was significantly (P less than 0.05) increased the percentage of MII oocytes as compared with 0 hr of culture. Cumulus expansion in the hormone experiments was 80% (none, bLH, and eLH), and 100% (eCG and FSH). Freshly prepared bLH significantly (P less than 0.05) inhibited nuclear maturation of equine oocytes. In summary, 15 hr of culture was sufficient time for equine oocyte IVM and all combinations of medium, serum, and hormone addition were equally effective in achieving IVM except fresh bLH and DM with BSA.     

Role of neurotransmitters and neuropeptides in the control of gonadotropin release: A review

Rapid progress has been recorded recently in the understanding of the role of neuro-transmitters and neuropeptides in the control of reproduction and on their apparent potential in the regulation of fertility. Peptides, as well as monoamines, are important in the control of lutinizing hormone releasing hormone and gonadotropin release. The input from brainstem noradrenergic neurons as well as dopamine mediated stimulated release of lutinizing hormone. In addition considerable evidence exist for the occurrence of a specific follicle stimulating hormone-releasing factor. A large number of brain peptides affect the secretion of lutinizing hormone releasing hormone and the endogenous opioid peptides appear to have a physiologically important function in restraining the influence on lutinizing hormone releasing hormone release under most circumstances. Vasoactive intestinal peptide and substanceP stimulate whereas cholecystokinin, neurotensin, gastrin, secretin, somatostatin α-melanosite stimulating hormone and vasotocin inhibit lutinizing hormone release. Of the inhibitory peptides, cholecystokinin and arg-vasotocin are the most potent. Inhibin injected into the ventricle selectively suppresses follicle stimulating hormone release by a hypothalamic action. Thus the control of gonadotropin release is complex and a number of aminergic and peptidergic transmitters are involved.     

Structural studies on equine chorionic gonadotropin

The amino acid sequence of the subunit of equine chorionic gonadotropin (eCG, also pregnant mare serum gonadotropin, PMSG) has been determined. Overlapping peptides from tryptic and chymotrypic digests were isolated by a two-dimensional peptide mapping technique and sequenced by the Edman procedure. The proposed amino acid sequence of eCG is: (**Denotes carbohydrate attachment points.) This sequence differs significantly from that proposed by Rathnamet al. (1978) for equine follitropin subunit; in particular, their sequence lacked the first fourteen residues.For the subunit we have placed in sequence 104 amino acid residues by direct sequence determination and peptide overlap procedures; in addition, 37 residues have been placed provisionally by homology with the human chorionic gonadotropin (hCG) sequence and composition and/or sequence data for the peptides isolated in the present studies. Difficulties in the procurement of the hormone have stalled completion of the -subunit amino acid sequence determination. The data now available indicate that eCG -subunit is highly homologous to hCG subunit and the subunits of luteinizing hormone from the pituitary gland of the several species so far described. The proposed partial sequence of eCG is:     

Cloning of partial putative gonadotropin hormone receptor sequence from fish

A search for the presence of mariner-like elements in the Labeo rohita genome by polymerase chain reaction led to the amplification of a partial DNA sequence coding for a putative transmembrane domain of gonadotropin hormone receptor. The amplified DNA sequence shows a high degree of homology to the available turkey and human luteinizing and follicle stimulating hormone receptor coding sequences. This is the first report on cloning such sequences of piscine origin.     

Superovulation in nonseasonal Japanese native goats, with special reference to the developmental progression of embryos

Treatment for superovulation with pregnant mare serum gonadotropin (PMSG) and follicle stimulating hormone (FSH) was carried out in nonseasonal breeder Japanese goats which are widely used as a substitute model for cattle in various studies in Japan. The proportion of females that came into estrus (93 and 99%) and the interval between PGF(2) administration and estrus (1.5 to 2.0 days) did not differ between females treated with PMSG and those treated with FSH. The number of normal embryos recovered was significantly higher (P<0.01) in FSH-treated (9.4 +/- 5.6) femals than in PMSG-treated females (5.7 +/- 4.4). The developmental stage of embryos recovered from 1.0 to 8.5 at 0.5-d intervals after mating is also described. The development to the two-cell, four-cell, eight-cell, morula, blastocyst and zona-free blastocyst stage was first observed 1.5, 2.5, 5.0 to 5.5, 6.0 and 6.5 d, respectively, after human chorionic gonadotropin (hCG) injection.     

Secretion of biologically active glycoforms of bovine follicle stimulating hormone in plants.

We chose the follicle stimulating hormone (FSH), a pituitary heterodimeric glycoprotein hormone, as a model to assess the ability of the plant cell to express a recombinant protein that requires extensive N-glycosylation for subunit folding and assembly, intracellular trafficking, signal transduction and circulatory stability. A tobacco mosaic virus (TMV) based transient expression system was used to express a single-chain (sc) version of bovine FSH in the tobacco related species Nicotiana benthamiana. Preparations of periplasmic proteins from plants infected with recombinant viral RNA contained high levels of sc-bFSH, up to 3% of total soluble proteins. Consistently, in situ indirect immunofluorescence revealed that the plant cell secreted the mammalian secretory protein to the extracellular compartment (EC). By mass spectrometric analysis of immunoaffinity purified sc-bFSH derived from EC fractions, we found two species of the plant paucimannosidic glycan type, truncated forms of complex-type N-glycans. Stimulation of cAMP production in a CHO cell line expressing the porcine FSH receptor acknowledged the native-like structure of sc-bFSH and a sufficient extent of N-glycosylation required for signal transduction. Furthermore, in superovulatory treatments of mice, sc-bFSH displayed significant in vivo bioactivity, although much lower than that of pregnant mare serum gonadotropin. We conclude that plants may have a broad utility as hosts for the recombinant expression of proteins even where glycosylation is essential for function.     

Studies on ovarian quiescence in the lactating bonnet monkey (Macaca radiata)

Lactating bonnet monkeys were used as a model to understand the mechanism of ovarian quiescence during lactation. The ovary of the bonnet monkey in the 3rd month of lactation responds well to exogenous pregnant mare serum gonadotropin stimulation with serum estrogen values reaching maximal levels by day 3 of the gonadotropin injection. The adminstration of ovine prolactin to such monkeys significantly inhibited the ovarian responsiveness to exogenous gonadotropin. The responsiveness of the pituitary of the lactating monkey (in the 3rd month of lactation) to luteinizing hormone releasing hormone injection was suppressed and supplementation with exogenous prolactin further accentuating this effect. The relative ability of chlorpromazine given intravenously/intramuscularly/intranasally to enhance endogenous prolactin levels was assessed. During the first 5 months of lactation when the basal prolactin levels were high, the luteinizing hormone levels were low. As the suckling stimulus reduces and prolactin levels fall, luteinizing hormone levels increase, the first post-parturient mensus occurring by 218 ± 4 days. This event was postponed by 3 months on increasing endogenous prolactin levels by administering chlorpromazine (250 μg/day by intranasal mode) over a 5 day period every month starting from the 3rd month of lactation.     

Peripheral administration of metastin induces marked gonadotropin release and ovulation in the rat

Metastin is a novel peptide that has been isolated from the human placenta as the cognate ligand of the G-protein-coupled receptor OT7T175 (or GPR54). However, its physiological functions have not yet been fully investigated. In the present study, we show that subcutaneous administration of metastin increased the plasma levels of gonadotropins (follicle-stimulating hormone and luteinizing hormone) and induced ovulation in prepubertal female rats that had been pretreated with pregnant mare serum gonadotropin to induce follicle maturation. Furthermore, metastin administration drastically increased the plasma levels of gonadotropins in male rats. This action was abolished by pretreatment with a GnRH antagonist, and was accompanied by induction of c-Fos immunoreactivity in GnRH neurons. These results suggest that s.c. administered metastin induces the release of gonadotropin via activation of the hypothalamic GnRH neurons.     

Induction and synchronization of estrus in dogs

Indications for estrus induction in the bitch include missed breeding opportunities or conception failure, the treatment of primary or secondary anestrus and synchronization of ovulation for embryo transfer programs. Reported methods for canine estrus induction include the use of synthetic estrogens (diethylstilbesterol), dopamine agonists (bromocryptine and cabergoline), GnRH agonists (lutrelin, buserelin, fertirelin, deslorelin, and leuprolide) and exogenous gonadotropins (luteinizing hormone, follicle stimulating hormone, human chorionic gonadotropin, pregnant mare serum gonadotropin, and human menopausal gonadotropin). These methods vary widely in efficacy of inducing estrus, as well as in the fertility of the induced estrus. The applicability of some of these methods for clinical practice is questionable. This review will summarize published reports on estrus induction and synchronization in bitches and summarize preliminary results using a long-acting injectable preparation of deslorelin.     

Effects of quinestrol on reproductive hormone expression, secretion, and receptor levels in female Mongolian gerbils (Meriones unguiculatus)

Quinestrol, a synthetic estrogen with marked estrogenic effects and prolonged activity, has potential as a contraceptive for Mongolian gerbils. The objective of this study was to describe the effects of quinestrol on reproductive hormone expression, secretion, and receptor levels in female Mongolian gerbils. Serum and pituitary concentrations of follicle stimulating hormone (FSH) and luteinizing hormone (LH) were decreased, whereas serum concentrations of estradiol (E2) and progesterone (P4) were increased after quinestrol treatment; the effects were both time- and dose-dependent. Furthermore, quinestrol downregulated expression of FSHβ and LHβ mRNA in the pituitary gland, as well as FSH receptor (FSHR) and estrogen receptor (ER) β in the ovary. However, it up-regulated mRNA expression levels of ERα and progesterone receptor (PR) in the pituitary gland and uterus, as well as mRNA for LH receptor (LHR) and PR in the ovary (these effects were time- and dose-dependent). In contrast, quinestrol had no significant effects on the mRNA expression levels of ERα in the ovary, or the gonadotropin α (GtHα) subunit in the pituitary gland. We inferred that quinestrol impaired synthesis and secretion of FSH and LH and that the predominant ER subtype in the pituitary gland of Mongolian gerbils may be ERα. Overall, quinestrol disrupted reproductive hormone receptor expression at the mRNA level in the pituitary-gonadal axis of the Mongolian gerbil.     

Serum chorionic gonadotropin levels determined by radioreceptorassay and early diagnosis of pregnancy in the cynomolgus monkey (Macaca fascicularis)

The radioreceptorassay developed to determine serum luteinizing hormone level in the cynomolgus monkey was evaluated for its usefulness in early pregnancy diagnosis by the detection of serum chorionic gonadotropin (CG). Blood samples were collected at weekly intervals from the 1st to the 5th week after conception to determine changes in circulating levels of CG. In the pregnancy cases, serum CG levels increased to above 50 μg/ml in almost all animals. By the determination of CG 3 weeks after conception, 86% of all pregnant cases exhibited a positive response. Cases that were negative 3 weeks after conception were followed by a repeated test in the next week. According to this test schedule, 95% of the pregnant cases were detected by 4 weeks after conception, and 5% were undetected as negative responses because their CG levels were low.     

Effect of super-ovulatory doses of pregnant mare serum gonadotropin and human chorionic gonadotropin in blastocyst implantation in golden hamsters

The effect of super-ovulatory dose of pregnant mare serum gonadotropin and human chorionic gonadotropin on ovulation, advancement of ovulation, subsequent embryo development and implantation were studied in the hamster. Groups of hamsters received pregnant mare serum gonadotropin injection on day 1 of the estrous cycle followed by human chorionic gonadotropin injection either at 56 or 76 h later, pregnant mare serum gonadotropin alone on day 1 or human chorionic gonadotropin alone on day 3.The combination therapy (pregnant mare serum gonadotropin and human chorionic gonadotropin) resulted in super-ovulation (an average of 40 mature ova/animal) while human chorionic gonadotropin alone yielded an average of 10 mature ova/animal. Ovulation was advanced by 24 h by giving human chorionic gonadotropin at 56 h instead of 76 h after pregnant mare serum gonadotropin. Subsequent embryo development and implantation occurring under different hormonal regimens were studied. The ova obtained by giving human chorionic gonadotropin injection at 56 h were poorly fertilizablein vivo and hence the pregnancy rate was low (6 %). These ova however, were fertilizablein vitro, suggesting that the low fertilization rate and developmental failure may be due to inhibition of sperm capacitation/transport because of premature human chorionic gonadotropin administration. In the group receiving human chorionic gonadotropin alone on day 3 there was fertilization and cleavage, but no implantation occurred due to failure of functional corpora lutea. However, administration of progesterone and estrone from day 2 of gestation resulted in 80% implantation and sustenance of pregnancy. On the other hand, the pregnant mare serum gonadotropin and human chorionic gonadotropin combination therapy resulted in super-pregnancy. The number of fetuses present at term was higher in the group receiving pregnant mare serum gonadotropin alone than in the group receiving the combination therapy. Embryo resorption however was higher (37%) in the latter group compared with the former (9.5%). However, preimplantation embryos were found to be viable as evidenced by fluorescein diacetate staining.     

Relative ability of ovine follicle stimulating hormone and itsβ-subunit to generate antibodies having bioneutralization potential in nonhuman primates

The relative ability of ovine follicle stimulating hormone and itsβ-subunit, two potential candidates for male contraceptive vaccine, to generate antibodies in monkeys capable of bioneutralizing follicle stimulating hormone was assessed usingin vitro model systems. Antiserum against native ovine follicle stimulating hormone was found to be highly specific to the intact form with no cross-reactivity with either of the two subunits while the antiserum againstβ-subunit of follicle stimulating hormone could bind to theβ-subunit in its free form as well as when it is combined withα-subunit to form the intact hormone. Both antisera could block the binding of the hormone to the receptor if the hormone was preincubated with the antibody. However, the follicle stimulating hormoneβ-antisera could only inhibit the binding of the hormone partially (33% inhibition) if the antibody and receptor were mixed prior to the addition of the hormone, while antisera to the native follicle stimulating hormone could block the binding completely (100% inhibition) in the same experiment. Similarly antisera to the native follicle stimulating hormone was significantly effective in blocking (100%) response to follicle stimulating hormone but not theβ-subunit antisera (0%) as checked using anin vitro granulosa cell system. Thus the probability of obtaining antibodies of greater bioneutralization potential is much higher if intact hormone is used as an antigen rather than itsβ-subunit as a vaccine. Majority of the work reported here was carried out during the tenure of Visiting Scientist fellowship awarded by the MRC Canada to the first author.     

Stimulation of the formation of 13,14-dihydroprostaglandin F2 alpha by gonadotropin in rat ovary

Effects of pregnant mare serum gonadotropin and human chorionic gonadotropin on the formation of 13,14-dihydroprostaglandin F2 alpha, a biologically active compound, were investigated in rat ovarian homogenate. The mass number of the compound, which was formed prostaglandin F2 alpha via 13,14-dihydro-15-ketoprostaglandin F2 alpha in rat ovarian homogenate but was not produced in rat homogenate, accorded with that of the authentic 13,14-dihydroprostaglandin F2 alpha by negative ion chemical ionization mass spectrometry. In the present experiment, the radioactivity of [3H]prostaglandin F2 alpha added to ovarian homogenate was decreased linearly and immediately until the incubation time of 10 min. The formation of 13,14-dihydroprostaglandin F2 alpha was increased up to 60 min. The formation of 13,14-dihydroprostaglandin F2 alpha from prostaglandin F2 alpha was markedly increased by pregnant mare serum gonadotropin and human chorionic gonadotropin. However, there was no additive or synergistic effect of these hormones. The formation of 13,14-dihydroprostaglandin F2 alpha from 13,14-dihydro-15-ketoprostaglandin F2 alpha weas also greatly stimulated by pregnant mare serum gonadotropin and human chorionic gonadotropin. The formation of 13,14-dihydro-15-ketoprostaglandin F2 alpha steeply declined until 24 h after treatment with human chorionic gonadotropin in pregnant mare serum gonadotropin-primed rats. In contrast, the formation of 13,14-dihydroprostaglandin F2 alpha was markedly increased until 24 h after human chorionic gonadotropin treatment, and the level was about 2.5-fold higher than that at 0 h, 48 h after injection of pregnant mare serum gonadotropin.     

Constant and variable regions in glycoprotein hormone beta subunit sequences: implications for receptor binding specificity.

A quantitative statistical analysis has confirmed the high degree of homology between human luteinizing hormone, follicle stimulating hormone, thyroid stimulating hormone and human chorionic gonadotropin beta subunit sequences, and has demonstrated much higher and extensive homology between follicle stimulating hormone and the others than was previously thought. Three “variable” zones have been detected in these sequences and these are likely to contain many of the residues responsible for the determination of receptor interaction specificity. For luteinizing hormone, comparison of sequences from different species has reduced the range of these residues to positions 1 to 6, 11, 14, 39 to 53, 75, 94, 97, 101, 104 and 108.     

Expression and regulation of SNAP-25 and synaptotagmin VII in developing mouse ovarian follicles via the FSH receptor

Soluble-NSF attachment protein receptor (SNARE) proteins play a role in vesicle fusion, exocytosis, and intracellular trafficking in neuronal cells as well as in fertilization and embryogenesis. We investigated the expression patterns of two SNARE proteins, SNAP-25 and synaptotagmin VII (SytVII), and their regulation by pregnant mare serum gonadotropin (PMSG) during mouse ovarian follicular development. Ovaries were obtained at 0, 12, 24, 36, and 48 h post-PMSG injection of immature mice. SNAP-25 and SytVII mRNA expression levels increased gradually in a time-dependant manner. However, protein levels revealed different patterns of expression, suggesting different translational regulation following PMSG stimulation. SNAP-25 and SytVII expression was closely associated with thickening of the granulosa cell (GC) layer and follicle morphological changes from a flattened to a cuboidal shape. To explore follicle stimulating hormone receptor (FSHR)-mediated regulation of their expression, GCs from preantral follicles were cultured to examine the effects of FSHR siRNA knockdown. FSHR siRNA abolished upregulation of the SNAREs in both PMSG and FSH-stimulated GCs. This abolished gene expression was rescued by adding dibutyryl cyclic AMP to the cultures. These results suggest that SNAP-25 and SytVII expression is regulated via the FSHR-cAMP pathway during follicular development.     

eFSH in clinical equine practice

Equine follicle stimulating hormone (eFSH) has been used to induce follicular development in transitional mares and problem acyclic mares, as well as superovulate cycling mares. The most efficacious protocol is to administer 12.5 mg eFSH, intramuscularly, twice daily beginning 5 to 7 days after ovulation when the diameter of the largest follicle is 20 to 25 mm. Prostaglandins are to be administered on the second day of eFSH therapy. Treatment with eFSH is continued for 3 to 5 days until follicle(s) are >or=35 mm in diameter. The mare is subsequently allowed to 'coast' for 36 h, after which human chorionic gonadotropin is administered to induce ovulation.     

Physicochemical and biological characterizations of pregnant mare serum gonadotropin and its subunits.

Pregnant mare serum gonadotropin and its subunits have been further characterized. Ultracentrifugation of the gonadotropin at pH 1.3 and 11.5 showed little evidence of dissociation compared to pH 8.2. Highly purified subunits are obtained by urea dissociation and ion-exchange chromatography followed by gel-filtration. Circular dichroism spectra of the gonadotropin and its subunits are much like those of ovine lutropin and its subunits in that there is little evidence for secondary structure and one or more tyrosine residues are inaccessible in the intact gonadotropin compared to the subunits. The alpha-subunit possesses almost 3 times as much total carbohydrate as the beta-subunit; the individual sugar composition of each was determined as well as the amino acid composition. The alpha-subunit begins with the sequence NH2-Phe-Pro (Gly or Pro) ... and terminates with isoleucine. The beta-subunit has the sequence NH2-Ser-Pro-Gly ...; no C-terminal residue is detectable by either carboxypeptidase or hydrazinolysis. Biological studies show the gonadotropin to be active in assays specific for both lutropin and follitropin. Precipitin test in agar with rabbit antiserum against the gonadotropin show that the beta subunit cross-reacts whereas the alpha subunit does not.     

Gonadotropin regulation of rat ovarian lysosomes: Existence of a hormone specific dual control mechanism

Gonadotropic hormones PMSG (15 IU/rat), FSH (3 g/rat), LH (9 g/rat) and hCG (3 g/rat) were shown to decrease the free cytosolic lysosomal enzymes during the acute phase of hormone action in rat ovaries. When isolated cells from such rats were analyzed for the cathepsin-D activity, the granulosa cells of the ovary showed a reduction in the free as well as in the total lysosomal enzyme activities in response to FSH/PMSG; the stromal and thecal compartment of the ovary showed a reduction only in the free activity in response to hCG/PMSG. The results suggest the presence of two distinct, target cell specific, mechanisms by which the lysosmal activity of the ovary is regulated by gonadotropins.Abbreviations PMSG pregnant mare serum gonadotropin - FSH follicle stimulating hormone - LH luteinizing hormone - hCG human chorionic gonadotropin - GC granulosa cells - S/T stromal and thecal cells