Two free isoforms of ovine glycoprotein hormone alpha-subunit strongly differ in their ability to stimulate prolactin release from foetal pituitaries.

alpha-Subunit dissociated from glycoprotein hormones has been previously shown to stimulate rat pituitary lactotroph differentiation and proliferation. However, whether the free form of the alpha-subunit (free alpha) can also play such a role is not known. To test whether free alpha may act on prolactin (PRL) release from ovine foetal pituitaries, this molecule was purified and two major isoforms, alphaA and alphaB were isolated. Free alphaA was found to be more acidic and more hydrophobic than both free alphaB and ovine LH alpha-subunit (oLHalpha). Free alphaA and oLHalpha exhibited a molecular mass of 14 kDa as determined by mass spectrometry, whereas free alphaB displayed a molecular mass of only 13.5 kDa because of its truncated N-terminus. All three alpha molecules bear mature-type N-linked saccharide chains including Nacetyl galactosamine residues but none of them contains O-linked oligosaccharide. The free alphaA isoform, more than the oLHalpha, was able to stimulate PRL release from ovine foetal pituitary explants in culture, whereas the free alphaB isoform displayed no activity. Moreover, the free alphaA and alphaB isoforms were able to recombine with the ovine LH beta-subunit (oLHbeta). The free alphaB/oLHbeta, and the oLHalpha/oLHbeta dimer were 4-fold more active than the free alphaA/oLHbeta dimer in a specific LH radioreceptor assay and in the stimulation of testosterone release from rat Leydig cells. The present study demonstrates that the two free alpha isoforms of ovine glycoprotein hormones exhibit distinct efficiencies in stimulating PRL release from ovine foetal pituitaries. Moreover, despite their identical ability to recombine with the oLHbeta, the free alpha isoform, which is the most efficient on PRL release, is the least efficient in conferring LH activity on the alpha/beta dimer.

Kinetic studies and production rate of equine (e) FSH in ovariectomized pony mares. Application to the determination of a dosage regimen for eFSH in a superovulation treatment.

The appropriate dosage regimen for equine FSH (eFSH) (dose, dosing interval) administration in a superovulation treatment in pony mares was determined by a kinetic approach using production rates and kinetic parameters of elimination of the hormone. Two dosage regimens were then tested in superovulation protocols. The eFSH production rates were determined by sampling four ovariectomized pony mares every 10 min for 8 h during the breeding season. Kinetic parameters were determined by administering four dose levels of a preparation of eFSH (4.4, 8.8, 17.6 and 35.2 micro g/kg) by the i.v. route to the same mares, in a randomized 4x4 Latin Square protocol. The overall mean plasma clearance was 0.256+/- 0.07 ml.kg(-1).min(-1), and was independent of the dose. The mean residence time ranged from 5.5 to 10.8 h and increased with the dose. The estimated FSH production rates were 8.6 to 15.3 micro g.kg(-1).day(-1) (i.e. 2.89 to 3.45 mg per day per mare). Two dosage regimens of eFSH were then tested in cyclic mares (ten treated mares in each trial): 3.45 mg per day (4.4 micro g/kg three times a day by the i.v. route), which corresponds to the maximal daily production rate of the native hormone in ovariectomized mares, and 1.72 mg per day (2.2 micro g/kg three times a day), which corresponds to half of that production rate. The dosage regimen of 2.2 micro g/kg three times a day gave satisfactory results in terms of efficacy (numbers of ovulations and embryos) with minimal unwanted effects (luteinized or anovulatory follicles).

Expression of a single betaalpha chain protein of equine LH/CG in milk of transgenic rabbits and its biological activity.

Equine chorionic gonadotropin (eCG) is a heavily glycosylated glycoprotein composed of non-covalently linked alpha- and beta-subunits. eCG possesses the particularity to bind to both LH and FSH receptors in species other than horses and to have a prolonged plasma half-life. All these properties make it of utmost interest for livestock fertilization program. Up to now, the only source of eCG is the serum of pregnant mare. Rabbit mammary gland is considered as a system able to produce recombinant glycoproteins in sufficient quantity for pharmaceutical use. Here we described the production of a recombinant single betaalpha chain of eLH/CG in the milk of transgenic rabbit. The construction of a single-chain permits to by-pass the problem of association-dissociation of the subunits. This recombinant hormone is greatly expressed (21.7 mg/l) and presents similar in vitro LH and FSH bioactivities. However, betaalphaeLH/CG shows an extremely rapid clearance (approximately 10 min), which could explain the absence of in vivo biological activity. So the rabbit mammary gland is not appropriate for the production of a recombinant active eLH/CG.

Ovine follicular fluid inhibits aromatase activity.

"Within follicle" regulations may be important for the fine tuning of gonadotrophin action in ovarian follicles. While numerous growth factors, steroids or proteins which are present in follicular fluid have been shown to have the ability of positively or negatively affecting follicle function, the net effect of follicular fluid of the dominant follicle on its function is unclear.A bioassay measuring aromatase activity of follicular walls was used (1) to check whether follicular fluid from dominant follicles can alter aromatase activity (2), to check how follicle size, atresia and specific gonadotrophins alter the effects of follicular fluid (3), to identify the nature (steroid or protein) of the active compound(s), and (4) to check whether the inhibition is specific of aromatase. Dominant follicular fluid had the ability to reduce aromatase activity. This effect was dose dependent and was obvious whether or not a protease inhibitor was added to the incubation medium. There was no difference in the magnitude of the inhibitory effect of follicular fluid when FSH (2 ng/ml) or no FSH was added to the incubation medium. LH, however, could potentialise the inhibitory effects of follicular fluid. Dominant follicular fluid was more potent to inhibit aromatase than follicular fluid from atretic follicles. Medium conditioned by granulosa cells, but not by theca cells could inhibit aromatase activity when added to the incubation medium. Charcoal treatment of dominant follicular fluid did not remove its inhibitory potential. Fractionation of dominant follicular fluid by a desalting column demonstrated that the inhibition was related to a compound(s) > 10 kDa. Finally, the effect of dominant follicular fluid on aromatase appears specific of this enzyme as follicular fluid does not affect androgen output by thecal shells or progesterone output by luteal cells. Further research is required to check whether the activity observed in dominant follicular fluid is related to compounds known to affect aromatase activity (inhibin, mullerian inhibiting substance, heat shock protein 90, superoxyde dismutase) or to another peptide/protein.

Deterioration of goat sperm viability in milk extenders is due to a bulbourethral 60-kilodalton glycoprotein with triglyceride lipase activity.

The aim of this work was to purify, identify, and characterize the component of goat bulbourethral gland secretion (BUS) responsible for goat sperm deterioration in skim milk extender. BUS extracts promote a decrease in the percentage of motile spermatozoa, deterioration in the quality of movement, breakage of acrosomes, and cellular death of goat spermatozoa diluted in skim milk. A 55- to 60-kDa monomeric glycoprotein (BUSgp60) was purified by cation-exchange, concanavalin A, and heparin-affinity chromatography and was identified as the only BUS component responsible for the deterioration of spermatozoa in milk. The BUSgp60 was shown to display triacylglycerol hydrolase activity, and partial sequences (37 amino acids in all) exhibited 50-70% homology with sequences of various types of pancreatic lipases (PLs), especially PL-related protein 2 (PL-RP2). In addition, porcine PL produced deterioration of goat sperm properties in milk as effectively as BUS and BUSgp60. These results support the preliminary identification of the goat BUSgp60 as a bulbourethral lipase belonging to the PL-RP2 family. Since seminal plasma also contains factors favorable for sperm survival, it is envisaged that, for the best preservation of goat semen, specific inhibitors of this family of lipase could be added into milk-based extenders without eliminating seminal plasma.

Inactivation of the olfactory amygdala prevents the endocrine response to male odour in anoestrus ewes.

Our aim was to study the role of the olfactory amygdala (medial and cortical nuclei) and the ventromedial nucleus of the hypothalamus (VMN) in the ability of the male odour or live males to induce a release of luteinizing hormone in anoestrus ewes. To achieve this, we temporarily blocked the activity of these structures by localized retrodialysis administration of the anaesthetic lidocaine. The effect of ram odour on the secretion of luteinizing hormone was completely blocked by inactivation of the cortical nucleus of the amygdala. In contrast, inactivation of part of the accessory olfactory system (the medial nucleus of the amygdala or the VMN) had no effect. In the presence of the male, lidocaine never impaired the endocrine response of the ewes. These results show that modulation of reproduction by the sexual partner even through pheromonal cues does not occur via the direct circuit of the accessory system. On the contrary, the cortical nucleus of the amygdala is absolutely necessary for the treatment of and/or the response to the male olfactory signal but this structure can be bypassed when other sensory cues are available.

Induction of ovulation and superovulation in mares using equine LH and FSH separated by hydrophobic interaction chromatography.

Pharmacological control of reproduction in mares requires the use of equine gonadotrophins to avoid induced immunological resistance. Crude equine gonadotrophins (CEG) have been used but the presence of equine luteinizing hormone (eLH) and follicle-stimulating hormone (eFSH) in CEG has led to disappointing results in superovulation studies. Separation of eLH and eFSH activities from CEG is necessary to overcome this problem. The hydrophobic properties of the two hormones were sufficiently different to permit their separation by hydrophobic interaction chromatography (HIC) on a phenyl Sepharose matrix. Good yields of separate FSH and LH fractions were readily obtained by stepwise elution and the method was adapted for large scale preparations of enriched fractions of eLH and eFSH. Two experiments were performed in vivo to evaluate the biological activity of the HIC fractions. Experiment 1 showed that biological activity of the LH fraction in inducing ovulation of preovulatory follicles was similar to that obtained with CEG, indicating that LH bioactivity was not altered by HIC. Experiment 2 demonstrated that biological activity of the FSH fraction was identical (as far as rate of ovulation was concerned) to that of CEG in superovulating mares, indicating that FSH activity was also not altered by HIC. Although we have not obtained better results with the separate equine gonadotrophins than with CEG, it is potentially advantageous to use preparations with single activity to obtain a controlled balance of FSH and LH activity. The HIC technique was chosen because it could easily be scaled up to provide the large amounts of the separate hormones needed for the treatment of a large number of mares.