Progesterone (P) powerfully inhibits gonadotropin-releasing hormone (GnRH) secretion in ewes, as in other species, but the neural mechanisms underlying this effect remain poorly understood. Using an estrogen (E)-free ovine model, we investigated the immediate GnRH and luteinizing hormone (LH) response to acute manipulations of circulating P concentrations and whether this response was mediated by the nuclear P receptor. Simultaneous hypophyseal portal and jugular blood samples were collected over 36 hr: 0-12 hr, in the presence of exogenous P (P treatment begun 8 days earlier); 12-24 hr, P implant removed; 24-36 hr, P implant reinserted. P removal caused a significant rapid increase in the GnRH pulse frequency, which was detectable within two pulses (175 min). P insertion suppressed the GnRH pulse frequency even faster: the effect detectable within one pulse (49 min). LH pulsatility was modulated identically. The next two experiments demonstrated that these effects of P are mediated by the nuclear P receptor since intracerebroventricularly infused P suppressed LH release but 3␣-hydroxy-5␣-pregnan-20-one, which operates through the type A ␥-aminobutyric acid receptor, was without effect and pretreatment with the P-receptor antagonist RU486 blocked the ability of P to inhibit LH. Our final study showed that P exerts its acute suppression of GnRH through an E-dependent system because the effects of P on LH secretion, lost after long-term E deprivation, are restored after 2 weeks of E treatment. Thus we demonstrate that P acutely inhibits GnRH through an E-dependent nuclear P-receptor system. Progesterone (P) is the dominant ovarian steroid present in the peripheral circulation during the mammalian reproductive cycle and serves a number of important regulatory roles. The luteal phase elevation in P inhibits pulsatile gonadotropinreleasing hormone (GnRH) and luteinizing hormone (LH) secretion (1-4) and prevents the occurrence of GnRH (5) and LH (6, 7) surges in response to fluctuations in peripheral estrogen (E) levels that accompany the waves of follicular growth occurring in the ovary (8). We have also recently demonstrated that the luteal phase elevation of P affects both the timing of the LH surge relative to E stimulation and the magnitude of the coincident GnRH surge (D.C.S., N.P.E., and A.C., unpublished results). Despite its obvious importance in regulating reproduction, little is known about how and where P acts to powerfully inhibit the neuroendocrine reproductive axis.Our first study, using the hypophyseal portal cannulation approach, assessed directly the timing of the changes in GnRH secretion that follow both an abrupt decrease and an increase in circulating P concentrations. To avoid potential P-E interactions, short-term ovariectomized (OVX) ewes were used.Since P modulates tonic LH secretion by affecting GnRH pulse frequency (1-4), it is held that P acts through neural targets and putative candidates include the opioidergic, noradrenergic, and ␥-aminobutyric acid (GABA) systems (9, 10). The p...

AMP-activated protein kinase (AMPK), a key regulator of cellular energy homeostasis, is present in metabolic tissues (muscle and liver) and has been identified as a modulator of the female reproductive functions. However, its function in the testis has not yet been clearly defined. We have investigated the potential role of AMPK in male reproduction by using transgenic mice lacking the activity of AMPK catalytic subunit α1 gene [α1AMPK knockout (KO)]. In the testis, the α1AMPK subunit is expressed in germ cells and also in somatic cells (Sertoli and Leydig cells). α1AMPK KO male mice show a decrease in fertility, despite no clear alteration in the testis morphology or sperm production. However, in α1AMPK(-/-) mice, we demonstrate that spermatozoa have structural abnormalities and are less motile than in control mice. These spermatozoa alterations are associated with a 50% decrease in mitochondrial activity, a 60% decrease in basal oxygen consumption, and morphological defects. The α1AMPK KO male mice had high androgen levels associated with a 5- and 3-fold increase in intratesticular cholesterol and testosterone concentrations, respectively. High concentrations of proteins involved in steroid production (3β-hydroxysteroid dehydrogenase, cytochrome steroid 17 alpha-hydroxylase/17,20 lysate, and steroidogenic acute regulatory protein) were also detected in α1AMPK(-/-) testes. In the pituitary, the LH and FSH concentrations tended to be lower in α1AMPK(-/-) male mice, probably due to the negative feedback of the high testosterone levels. These results suggest that total α1AMPK deficiency in male mice affects androgen production and quality of spermatozoa, leading to a decrease in fertility.

Although this culture model is clearly unsuitable for preparing germ cells for therapeutic purposes, it does represent a most valuable tool for testing the effects of biological and chemical agents on testicular tissue.

The mechanism of fertilization remains largely enigmatic in mammals. Most studies exploring the molecular mechanism underlying fertilization have been restricted to a single species, generally the mouse, without a comparative approach. However, the identification of divergences between species could allow us to highlight key components in the mechanism of fertilization. In the pig, in vitro fertilization (IVF) and polyspermy rates are high, and spermatozoa penetrate easily through the zona pellucida (ZP). In contrast, IVF rates are low in the horse, and polyspermy is scarce. Our objective was to develop a comparative strategy between these two divergent models. First, we compared the role of equine and porcine gametes in the following five functions using intraspecific and interspecific IVF: ZP binding, acrosome reaction, penetration through the ZP, gamete fusion, and pronucleus formation. Under in vitro conditions, we showed that the ZP is a determining element in sperm-ZP attachment and penetration, whereas the capacity of the spermatozoa is of less importance. In contrast, the capacity of the spermatozoa is a key component of the acrosome reaction step. Second, we compared the composition and structure of the equine and porcine ZP. We observed differences in the number and localization of the ZP glycoproteins and in the mesh-like structure of the ZP between equine and porcine species. These differences might correlate with the differences in spermatozoal attachment and penetration rates. In conclusion, our comparative approach allows us to identify determining elements in the mechanism of fertilization.

Estrogen exerts important feedback effects upon the biosynthetic and secretory behavior of gonadotropin-releasing hormone (GnRH) neurons to control reproductive functioning. The mechanism of estrogen action upon these neurons is unclear and seems likely to involve the transsynaptic regulation of GnRH neurons. The objective of the present study was to identify the estrogen-receptive neural populations which project to the general vicinity of the GnRH perikarya in the rostral preoptic area and diagonal band of Broca (rPOA/DBB) of the ewe. Intact breeding-season ewes received an injection of the retrograde tracer fluorogold (FG) into the rPOA/DBB, and their hypothalami and brainstems examined for the presence of FG and estrogen receptor α (ERα) immunocytochemistry. Retrogradely labeled neurons were identified principally within the lateral septum (LS), lamina terminalis, bed nucleus of the stria terminalis, POA, arcuate nucleus (ARN), ventromedial nucleus (VMN) and median eminence. Smaller numbers of FG-immonoreactive cells were found in the caudal brainstem where they resided mostly in the ventrolateral medulla (VLM). Dual-labeled cells exhibiting both FG and ERα staining were prominent in the POA, LS and at all rostrocaudal levels of the VMN and ARN. Small numbers of dual-labeled cells were found in the VLM. These observations indicate that a number of distinct ERα-expressing neural populations project to the rPOA/DBB where the majority of the GnRH perikarya are found in the ewe. Although it is not possible to determine the direct connectivity of these projections with GnRH neurons, the findings provide an initial neuroanatomical framework through which the transsynaptic actions of estrogen on ovine GnRH neurons may be tested.

Noradrenergic neurons are implicated in the estrogen-dependent neural regulation of luteinizing hormone secretion in a variety of mammalian species. The current study has used immunocytochemical methods to determine whether estrogen receptors (ER) are expressed within the brainstem of the ewe and to establish their relationship to noradrenergic neurons. Using a monoclonal mouse antiserum directed against the N-terminal of ERα, four distinct populations of ERα-immunoreactive cells were identified in ovine medulla and pons. The largest population was found in the superficial laminae of the spinal nucleus of the trigeminal nerve, followed by the nucleus tractus solitarius, lateral area postrema, and ventrolateral medulla. Double-labelling immunocytochemistry using antisera directed against the ERα and dopamine-β-hydroxylase revealed that noradrenergic neurons expressing ER immunoreactivity were only found in ventrolateral medulla (A1 cell group) and nucleus tractus solitarius (A2 cell group). No double-labelled cells were identified in the A5, A6, or A7 noradrenergic cell groups. ERs were expressed with a clear rostrocaudal topography within the A1 and A2 populations, with 80–90% of noradrenergic neurons expressing ERα in the caudalmost medulla as compared with less than 5% rostral to the obex. Our findings demonstrate that, as in the rat, the ovine A1 and A2 neurons express ERs in a defined topographical manner, while, dissimilar to the rat, ERα is not synthesized by noradrenergic neurons in the other cell groups. These observations indicate that A1 and A2 noradrenergic neurons in the ovine brainstem are likely to be influenced by circulating estrogens and lay the neuroanatomical foundations for investigating the functional role of these cell populations within the gonadotropin-releasing hormone neuron network of the sheep.

Adult transgenic mice overexpressing human insulin-like growth factor-binding protein-1 in the liver present reproductive abnormalities in both sexes. In the present work, we have investigated the mechanisms responsible for limiting breeding capacity in these transgenic male mice. Homozygous adult transgenic male mice (3-6 months old) exhibited irregular copulatory behavior and a reduction of the number of pregnancies per female as well as of litter size per pregnancy. Genital tract weight, more specifically epididymal and seminal vesicle weights, were reduced by 45% in homozygous transgenic vs. nontransgenic mice. Homozygous transgenic mice exhibited a 30% reduction of the length of seminiferous tubules (P = 0.007), a 30% decrease in daily sperm production per testis (P = 0.019), and a 50% decrease in the number of spermatozoa in testis (P = 0.037), associated with morphological abnormalities of the sperm heads leading to an approximately 50% reduction of fertilized two-cell eggs (P = 0.002) and of implanted embryos on d 5.5 after mating (P = 0.004). The round spermatids also appeared altered in their morphology. In addition, Leydig cells in homozygous transgenic mice exhibited an altered appearance, with a 1.8-fold increase in lipid droplets in their cytoplasm (P < 0.001). Moreover, the concentration of 3beta-hydroxysteroid dehydrogenase was 66% lower in testis from transgenics compared with those from normal mice (P = 0.01), leading to a tendency toward lower plasma testosterone levels (P = 0.1). Interestingly, LH concentrations were increased by 40% in transgenic pituitary extracts (P = 0.02), and basal LH secretion by pituitary explants in vitro was increased by 60% in homozygous transgenic vs. normal mice (P = 0.04), suggesting an alteration of LH pulsatile secretion in vivo. In conclusion, these data suggest that the breeding impairment of human insulin-like growth factor-binding protein-1 transgenic males is due at least in part to an alteration of the process of spermatogenesis, leading to a diminution of sperm production and of its quality. Minor impairment of steroidogenesis may also contribute to the reduced reproductive capacity of these animals. Our observations are consistent with the idea that normal spermatogenesis and perhaps also steroidogenesis are dependent on the actions of sufficient concentrations of unbound IGF-I.

Corticotrophin-releasing hormone (CRH) has been proposed as a mediator of the antireproductive effects of stress through an action within the hypothalamus to inhibit GnRH secretion. This hypothesis was tested in sheep by studying the responses to central administration of CRH in both sexes and in both seasons. Sexually mature, Ile-de-France ewes and Romanov rams that had been gonadectomized and implanted with a permanent guide cannula into the third cerebral ventricle were used. Ewes were studied in the presence and absence of exogenous oestradiol plus progesterone, in both the breeding and anoestrous seasons. All rams were treated with testosterone and were studied only during the breeding season. Each observation involved serial samples (every 10 min) of jugular blood for 5 h before (control) and 5 h after an intracerebroventricular (i.c.v.) injection of either saline (vehicle) or 5 nmoles CRH in 20 microliters vehicle. The saline injections did not affect any of the endocrine variables measured; however, CRH always increased cortisol concentrations in jugular plasma. In the absence of treatment with replacement sex steroids, icv injection of CRH had no effect on pulsatile LH secretion in females either during the breeding season or during anoestrus. However, LH pulse frequency and mean LH concentrations increased significantly on every occasion on which animals were treated with sex steroids. Treatment with CRH also increased LH secretion in the testosterone-treated rams. It is concluded that, contrary to the hypothesized role of CRH as an inhibitor of reproductive activity, this neuropeptide stimulates pulsatile LH (and thus GnRH) secretion, at least in this species. The fact that gonadal steroids seem to be obligatory for the expression of this effect suggests that the protocols used in past studies need to be reassessed.