Alan S. McNeilly

Detection of dimeric inhibin throughout the human menstrual cycle by two‐site enzyme immunoassay

OBJECTIVE We have developed and validated a two‐site immunoassay for the measurement of dimeric inhibin in plasma and subsequently measured dimeric inhibin levels in plasma through the normal female menstrual cycle.

PULSATILE SECRETION OF LH, FSH, PROLACTIN, OESTRADIOL AND PROGESTERONE DURING THE HUMAN MENSTRUAL CYCLE

The pulsatile secretion of gonadotrophins and ovarian steroids was studied in normal women at different stages of the menstrual cycle. The concentration of LH, FSH, Prolactin (PRL), oestradiol and progesterone were measured in samples of plasma collected every 15 min for 6 h and the frequency and amplitude of each episodic pulse of hormone estimated. Although significant fluctuations occurred in the concentration of each hormone, LH showed the most easily identifiable pulses the frequency of which increased significantly from the early follicular to the late follicular phase of the cycle (3.4±0.3 v. 4.4±0.2 pulses 6 h P<0.01). During the luteal phase the basal concentration of LH (5.6±0.9 U/l), pulse amplitude (7.4±1.7 U/l) and frequency (1.6±0.2/6 h) were much lower than at any stage of the follicular phase (P<0.001). The concentration of FSH and PRL showed a similar but less marked change to that of LH throughout the menstrual cycle with a significant decline in both basal concentration and pulse frequency in the luteal phase of the cycle. Although only 47% of all LH pulses were associated with a pulse of FSH, 70% of FSH and prolactin pulses occurred within 15 min of an LH pulse.

Theca: the forgotten cell of the ovarian follicle

Theca cells function in a diverse range of necessary roles during folliculogenesis; to synthesize androgens, provide crosstalk with granulosa cells and oocytes during development, and provide structural support of the growing follicle as it progresses through the developmental stages to produce a mature and fertilizable oocyte. Thecal cells are thought to be recruited from surrounding stromal tissue by factors secreted from an activated primary follicle. The precise origin and identity of these recruiting factors are currently not clear, but it appears that thecal recruitment and/or differentiation involves not just one signal, but a complex and tightly controlled combination of multiple factors. It is clear that thecal cells are fundamental for follicular growth, providing all the androgens required by the developing follicle(s) for conversion into estrogens by the granulosa cells. Their function is enabled through the establishment of a vascular system providing communication with the pituitary axis throughout the reproductive cycle, and delivering essential nutrients to these highly active cells. During development, the majority of follicles undergo atresia, and the theca cells are often the final follicular cell type to die. For those follicles that do ovulate, the theca cells then undergo hormone-dependent differentiation into luteinized thecal cells of the corpus luteum. While the theca is an essential component of follicle development and ovulation, we do not yet fully understand the control of recruitment and function of theca cells, an important consideration since their function appears to be altered in certain causes of infertility.

Release of oxytocin and prolactin in response to suckling.

The oxytocin and prolactin responses to suckling were measured in 10 women in early (n = 5) and established lactation (n = 5). Oxytocin was released in a pulsatile manner during suckling in all women, but the response was not related to milk volume, prolactin response, or parity of the mother. In all 10 women plasma oxytocin concentrations increased three to 10 minutes before suckling began. In five women this occurred in response to the baby crying, in three it coincided with the baby becoming restless in expectation of the feed, while in two it corresponded with the mother preparing for the feed. There was no prolactin response to stimuli other than stimulation of the nipple associated with suckling. These results clearly indicate that the milk ejection reflex, with release of oxytocin, occurs in most women before the tactile stimulus of suckling. A second release of oxytocin follows in response to the suckling stimulus itself. Thus it is important that care is taken to protect breast feeding mothers from stress not only during suckling but also immediately before nursing, when conditioned releases of oxytocin will occur.

Regulation of folliculogenesis and the determination of ovulation rate in ruminants.

The paper presents an update of our 1993 model of ovarian follicular development in ruminants, based on knowledge gained from the past 15 years of research. The model addresses the sequence of events from follicular formation in fetal life, through the successive waves of follicular growth and atresia, culminating with the emergence of ovulatory follicles during reproductive cycles. The original concept of five developmental classes of follicles, defined primarily by their responses to gonadotrophins, is retained: primordial, committed, gonadotrophin-responsive, gonadotrophin-dependent and ovulatory follicles. The updated model has more extensive integration of the morphological, molecular and cellular events during folliculogenesis with systemic events in the whole animal. It also incorporates knowledge on factors that influence oocyte quality and the critical roles of the oocyte in regulating follicular development and ovulation rate. The original hypothetical mechanisms determining ovulation rate are retained but with some refinements; the enhanced viability of gonadotrophin-dependent follicles and increases in the number of gonadotrophin-responsive follicles by increases in the throughput of follicles to this stage of growth. Finally, we reexamine how these two mechanisms, which are thought not to be mutually exclusive, appear to account for most of the known genetic and environmental effects on ovulation rate.

Effect of supplementary food on suckling patterns and ovarian activity during lactation.

Patterns of infant feeding, basal prolactin concentrations, and ovarian activity were studied longitudinally in 27 breast-feeding mothers from delivery until first ovulation. Suckling frequency (6.1 feeds/day) and suckling duration (122 mins/day) reached peak values four weeks post partum and remained relatively constant until the introduction of supplementary food at a mean of 16 weeks post partum. There were subsequently sharp declines in both the frequency and duration of suckling, both of which correlated closely with basal prolactin concentrations. None of the 27 mothers ovulated during unsupplemented breast-feeding, but within 16 weeks of introducing supplements ovarian follicular development had returned in 20 and ovulation in 14 mothers. The mothers who ovulated within 16 weeks of giving supplements had reduced frequency and duration of suckling more quickly and weaned more abruptly than those who continued to suppress ovulation. These data suggest that the introduction of supplementary food may exert an important and hitherto unrecognised effect on the timing of first ovulation by reducing the frequency and duration of suckling episodes.

Cloning and sequencing of the sheep pituitary gonadotropin-releasing hormone receptor and changes in expression of its mRNA during the estrous cycle

We have isolated a full length cDNA clone coding for the sheep GnRH receptor (GnRH-R). The amino acid sequence shows greater homology to the human GnRH-R sequence than the two rodent receptors published so far. We have also carried out physiological studies investigating the pattern of expression of the GnRH-R mRNA throughout the estrous cycle. GnRH receptor mRNA and GnRH binding levels were both significantly (P < 0.05) increased over luteal levels up until the time of the preovulatory LH surge, whilst post-surge, a significant (P < 0.05) decline was seen. These changes were related to increased follicle estradiol production in the follicular phase. In contrast, no changes in the abundance of LH beta mRNA were seen throughout the estrous cycle, but the pituitary content of LH significantly (P < 0.05) decreased after the preovulatory LH surge. These results suggest that there is a close relationship between the abundance of GnRH-R mRNA and translation of the GnRH-R in sheep.

Homozygosity for a single base-pair mutation in the oocyte-specific GDF9 gene results in sterility in Thoka sheep

The control of fecundity is critical in determining mammalian offspring survival. It is regulated principally by the ovulation rate, so that primates and large farm species commonly have a single offspring. Previously, several mutations have been identified in sheep which increase the naturally low ovulation rate; although in some cases homozygous ewes are infertile. In the present study we present a detailed characterization of a novel mutation in growth differentiation factor 9 (GDF9), found in Icelandic Thoka sheep. This mutation is a single base change (A1279C) resulting in a nonconservative amino acid change (S109R) in the C-terminus of the mature GDF9 protein, which is normally expressed in oocytes at all stages of development. Genotyping all animals for which reproductive records were available confirmed this mutation to be associated with increased fecundity in heterozygous ewes and infertility in homozygotes. Analysis of homozygote ovarian morphology and a number of genes normally activated in growing follicles showed that GDF9 was not involved in oocyte activation, but in subsequent development of the follicle. This study highlights the importance of oocyte factors in regulating fertility and provides new information for structural analysis and investigation of the potentially important sites of dimerization or translational modifications required to produce biologically active GDF9. It also provides the basis for the utilization of these animals to enhance sheep production.

Multiple factors affecting the efficiency of multiple ovulation and embryo transfer in sheep and goats

This review offers an overview of the basic characteristics of in vivo embryo technologies, their current status, the main findings and the advances gained in recent years, and the outstanding subjects for increasing their efficiency. The use of superovulation and embryo transfer procedures remains affected by a high variability in the ovulatory response to hormonal treatment and by a low and variable number of transferable embryos and offspring obtained. This variability has been classically identified with both extrinsic (source, purity of gonadotrophins and protocol of administration) and intrinsic factors (breed, age, nutrition and reproductive status), which are reviewed in this paper. However, emerging data indicate that the main causes of variability are related to endocrine and ovarian factors, and so the number of studies and procedures addressing a better understanding and control of these factors may be increased in the future. The accomplishment of this objective, the improvement of procedures for embryo conservation and for the selection and management of recipient females, will allow further development and application of this technology.

Hypothalamic pulse generators

Publisher Summary This chapter describes pulsatile hormone secretion and analyzes the mechanisms underlying the neural organization of this phenomenon in context of oxytocin and luteinizing hormone releasing hormone (LHRH) secretion. It also discusses five concepts— (1) oxytocin and LHRH are both released from the hypothalamus in pulses superimposed upon a continuous or intermittent low level of secretion; these two modes of secretion can produce separate actions, or one might govern the response to the other; (2) the synchronous generation of action potentials at a very fast rate within a population of peptidergic neurones provides the neural substrate for the release of a hormone pulse; (3) the control of interpulse interval is determined by events within the brain, though the interval generator may not reside within the neurons that secrete oxytocin or LHRH; (4) amplitude modulation of pulsatile secretion can relate to different levels of electrical activation within the brain and/or to differences in the responsiveness of the target tissues that transduce the pulsatile signal; and (5) opioid peptides inhibit the secretion of both oxytocin and LHRH. This involves an inhibition of stimulus-secretion coupling within the nerve terminals, and possibly an inhibition of synaptically mediated events that impinge upon the cell bodies of the peptidergic neurons within the hypothalamus.