Equine luteinizing hormone : measurement and regulation of serum levels

Abstract

An in vitro bioassay based on LH-stimulated testosterone production by dispersed mouse Leydig cells was validated for use in the horse. Using this bioassay and a previously validated heterologous radioimmunoassay (RIA), it was found that the patterns of LH levels measured by the 2 assay methods during the mare's oestrous cycle were similar but not identical, so that the ratio of biological:immunological (B:I) activity changed significantly during the cycle; being high as LH levels rose during oestrus but falling sharply on the last day of oestrus to remain stable and low through dioestrus. Investigating factors affecting the B:I ratio of serum LH, it was observed that the low ratio in serum from seasonally acyclic mares could be raised by pre-treatment with oestradiol, followed by pulse injection of gonadotrophin-releasing hormone (GnRH). Neither GnRH nor oestradiol treatment alone could alter B:I ratio in acyclic mare serum. Thus, it was suggested on the basis of this observation and review of relevant literature that the rising serum oestradiol levels preceding ovulation and increased GnRH levels which may occur at this time could contribute to the presence in serum of LH forms with greater biological potency. Changes in B:I ratio during the oestrous cycle implied qualitative differences in the circulating molecule. The nature and source of these differences were investigated by using the technique of isoelectric focussing (IEF) to separate, on the basis of charge, the various forms of LH in pituitary extracts and serum samples. Isoelectric focussing of pituitary extracts resulted in LH peaks at pH 7.2, 6.1, 5.2 and 4.5, with differring B:I ratio; mean ratio being greatest in the pI 5.2 peak and least in the 7.2 peak. When horse serum was focussed, marked similarities between serum and pituitary LH were observed with peaks of LH activity occurring at like pI values and B:I ratio changing similarly with the pI of peaks. Serum LH was different to pituitary LH in that immunoactivity without bioactivity was found at either pH extreme. When IEF profiles of high and low B:I ratio sera (paired samples from 2 cyclic mares) were compared, no consistent shift in LH distribution was observed with decreased ratio; however, in both low ratio sera, relatively more LH activity was found in areas of low B:I ratio and consequently less LH activity was recovered in the highest ratio peak. While interpretation of these results was complicated by the small number of samples focussed, it appeared that changes in the B:I ratio of serum LH could be related to structural changes in the circulating molecule. Whether the polymorphism of serum LH originated entirely from the pituitary or in part from post-secretory modification of the molecule could not be conclusively answered. The close correspondence between pI values at which the bulk of pituitary and serum LH focussed attested to the pituitary being the major source of this polymorphism; however the presence in serum but not pituitary of LH with little or no bioactivity suggested that some post-secretory mutation might occur. This question was raised again in Section 3, when little correlation could be found between patterns of bio- and immuno-active LH levels in serum samples collected at 5 or 15-20 min intervals from cyclic mares. In oestrous mares, rapid, low amplitude pulses in serum LH levels were shown to occur with statistically significant regularity when measured by bioassay but not by radioimmunoassay. Results with dioestrous\mares were highly variable with pulses similar in amplitude and frequency to those observed at oestrus demonstrable in some, but not all, mares. In dioestrus mares in which LH pulses could not be shown and in seasonally acyclic mares, serum LH levels remained stable and did not appear to decay during the period of observation regardless of assay method.

The physiological significance of the above observations requ1res further investigation; nevertheless, the fact that the mare ovulates when relative biopotency of serum LH is maximal as assessed by mouse Leydig cells, suggests both that qualitative changes in the circulating molecule do have physiological importance and that the response of equine target cells can be predicted adequately by the mouse (a supposition supported by preliminary results from an in vitro bioassay using horse Leydig cells). The practical significance of these findings is that there appear to be circumstances in which the radioimmunoassay used here would not give adequate information on the level of LH stimulation at target tissues. Furthermore, it is evident that bio- and immunoassay measure forms of the hormone which, especially at oestrus, appear in serum with different time courses.

In the course of experiments in which GnRH was given to mares to determine the effect of stimulation on the nature of the circulating LH molecule it was observed that both bio- and immuno-active LH responses to a small dose of GnRH (i.e. "pituitary responsiveness") were lower in early oestrus than in early dioestrus, whereas pre-injection LH levels were higher in oestrus than dioestrus. Furthermore, additional experiments demonstrated that in the mare the ovulatory LH surge began without a corresponding increase in pituitary responsiveness to GnRH stimulation suggesting that increased GnRH input to the pituitary was responsible for the onset of the LH surge. The basis for this deduction was proved to be less than firm when subsequent experiments failed to demonstrate a GnRH dose-LH response relationship in oestrous mares, with GnRH doses ranging from 0.05 mg to 2.0 mg eliciting similar responses. In the absence of a GnRH dose- LH response relationship, estimates of pituitary responsiveness (i.e. LH release/unit GnRH) ,would vary with GnRH dose given, and therefore the response of the pituitary to endogenous GnRH input could not be inferred from response to exogenous GnRH administration. Thus, a GnRH dose-LH response relationship must be shown in oestrous mares before the relative importance of changes in pituitary responsiveness and endogenous GnRH secretion in producing the ovulatory LH surge can be confidently assessed.

In a series of experiments, the regulation of seasonal patterns of LH secretion in the male horse was studied. In particular, the importance of the testes in maintaining the annual pattern of serum LH levels was investigated by measuring LH levels in blood samples collected at approximately fortnightly intervals for a year from 5 “long-term” geldings (castrated>3 years). In these horses, no significant effect of month on LH levels was observed. By contrast, earlier work in our laboratory had shown that stallions in the same environment during one year have a markedly seasonal pattern of LH secretion, with LH levels rising at the onset of the breeding season to reach levels in late spring 3-4 times those in early winter. Despite differences in seasonal patterns of secretion, annual mean LH levels in geldings and stallions were similar. In mid-summer, serum LH levels were measured in 5 geldings castrated 4-6 weeks previously, 24 geldings castrated 3-25 years (in ng/ml) previously and 5 stallions; mean LH levels ± S.E.M. in (ng/ml) were: 63.5 ± 8.8, 14.0 ± 1.6 and 1.5 respectively. In the long-term geldings LH levels were not affected by age, indicating that following the initial postcastration rise, LH levels fell to within the normal range of pre-castration values and stabilized.

These results show that the testes are necessary for maintenance of: the normal seasonal pattern of LH secretion in the male horse, including the increase in LH at the onset of the breeding season. Thus, it is possible that the effects of testicular hormones on LH secretion in the male horse may include not only a negative feedback at the hypothalamus/pituitary as observed in other species but also a positive component in the pathway by which LH is increased at the onset of the breeding season.