Progesterone receptor-Src kinase signaling pathway mediates neuroprogesterone induction of the luteinizing hormone surge in female rats.

Neural circuits in female rats are exposed to sequential estradiol and progesterone to regulate the release of luteinizing hormone (LH) and ultimately ovulation. Estradiol induces progesterone receptors (PGRs) in anteroventral periventricular nucleus (AVPV) kisspeptin neurons, and as estradiol reaches peak concentrations, neuroprogesterone (neuroP) synthesis is induced in hypothalamic astrocytes. This local neuroP signals to PGRs expressed in kisspeptin neurons to trigger the LH surge. We tested the hypothesis that neuroP-PGR signaling through Src family kinase (Src) underlies the LH surge. As observed in vitro, PGR and Src are co-expressed in AVPV neurons. Estradiol treatment increased the number of PGR immunopositive cells and PGR and Src colocalization. Furthermore, estradiol treatment increased the number of AVPV cells that had extranuclear PGR and Src in close proximity (< 40 nm). Infusion of the Src inhibitor (PP2) into the AVPV region of ovariectomized/adrenalectomized (ovx/adx) rats attenuated the LH surge in trunk blood collected 53 h post-estradiol (50 µg) injection that induced neuroP synthesis. Although PP2 reduced the LH surge in estradiol benzoate treated ovx/adx rats, activation of either AVPV PGR or Src in 2 µg estradiol-primed animals significantly elevated LH concentrations compared to dimethyl sulfoxide infused rats. Finally, antagonism of either AVPV PGR or Src blocked the ability of PGR or Src activation to induce an LH surge in estradiol-primed ovx/adx rats. These results indicate that neuroP, which triggers the LH surge, signals through an extranuclear PGR-Src signaling pathway.

Luteinizing hormone-independent rise of progesterone as the physiological trigger of the ovulatory gonadotropins surge in the human.

The current ovarian cycle paradigm postulates that ovulation is triggered by a critically sustained elevation of estradiol. However, an in-depth look into the published data reveals considerable uncertainty about the relative roles of progesterone and estradiol in the ovulation process.This review provides compelling evidences that the role of estradiol in ovulation has been misinterpreted and that the true physiological trigger of ovulation is a luteinizing hormone-independent preovulatory progesterone surge in the circulation to approximately 0.5 ng/mL. Furthermore, the current work reconciles the ability of progesterone to trigger ovulation, with its well-established ability to block ovulation during pregnancy, or when administered in the form of a synthetic progestin in birth control formulations and with experimental data that estradiol benzoate triggers ovulation in the complete absence of progesterone.

Endocrinology of the Menopause.

In women, age-related changes in ovarian function begin in the mid-30s with decreased fertility and compensatory hormonal changes in the hypothalamus-pituitary-gonadal axis that maintain follicle development and estrogen secretion in the face of a waning pool of ovarian follicles. The menopause transition is characterized by marked variability in follicle development, ovulation, bleeding patterns, and symptoms of hyper- and hypoestrogenism. The menopause, which is clinically defined by the last menstrual period, is followed by the consistent absence of ovarian secretion of estradiol.

Characterizing the neuroendocrine and ovarian defects of androgen receptor-knockout female mice.

Homozygous androgen receptor (AR)-knockout (ARKO) female mice are subfertile due to both intra- and extraovarian (neuroendocrine) defects as defined by ovary transplantation. Using ARKO mice, this study set out to reveal the precise AR-regulated pathways required for optimal androgen-regulated ovulation and fertility. ARKO females exhibit deficient neuroendocrine negative feedback, with a reduced serum luteinizing hormone (LH) response to ovariectomy (OVX) (P < 0.01). Positive feedback is also altered as intact ARKO females, at late proestrus, exhibit an often mistimed endogenous ovulatory LH surge. Furthermore, at late proestrus, intact ARKO females display diminished preovulatory serum estradiol (E2; P < 0.01) and LH (P < 0.05) surge levels and reduced Kiss1 mRNA expression in the anteroventral periventricular nucleus (P < 0.01) compared with controls. However, this reduced ovulatory LH response in intact ARKO females can be rescued by OVX and E2 priming or treatment with endogenous GnRH. These findings reveal that AR regulates the negative feedback response to E2, E2-positive feedback is compromised in ARKO mice, and AR-regulated negative and positive steroidal feedback pathways impact on intrahypothalamic control of the kisspeptin/GnRH/LH cascade. In addition, intraovarian AR-regulated pathways controlling antral to preovulatory follicle dynamics are disrupted because adult ARKO ovaries collected at proestrus have small antral follicles with reduced oocyte/follicle diameter ratios (P < 0.01) and increased proportions of unhealthy large antral follicles (P < 0.05) compared with controls. As a consequence of aberrant follicular growth patterns, proestrus ARKO ovaries also exhibit fewer preovulatory follicle (P < 0.05) and corpora lutea numbers (P < 0.01). However, embryo development to the blastocyst stage is unchanged in ARKO females, and hence, the subfertility is a consequence of reduced ovulations and not altered embryo quality. These findings reveal that the AR has a functional role in neuroendocrine regulation and timing of the ovulatory LH surge as well as antral/preovulatory follicle development.

Effect of supplementation with different fat sources on the mechanisms involved in reproductive performance in lactating dairy cattle.

Supplementary fat positively influences reproductive performance in dairy cattle, although the mechanisms involved are not clearly defined. Our objective was to determine the effects of four different fat supplements on follicle development, plasma steroid hormone concentrations and prostaglandin (PG) synthesis in lactating dairy cattle. Forty-eight early lactation Holstein-Friesian cows (21 primiparous, 27 multiparous) were used in a completely randomized block design. Cows were fed the same basal TMR diet and received one of four fat supplements: (i) palmitic acid (18:0 fatty acid; Control), (ii) flaxseed (rich in 18:3 n-3 fatty acid; Flax), (iii) conjugated linoleic acid (a mixture of cis-9, trans-11 and trans-10, cis-12 isomers; CLA), and (iv) fish oil (rich in 20:5 and 22:6 n-3 fatty acids; FO). All lipid supplements were formulated to be isolipidic; palmitic acid was added as necessary to provide a total lipid supplement intake of 500 g/day. Cows were synchronized to be in estrus on Day 15 of dietary treatment. All antral follicles were counted, and dominant follicles, subordinate follicles and corpora lutea were measured daily via transrectal ovarian ultrasonography for one complete estrous cycle. Blood samples were collected daily, and selected samples were analyzed for progesterone, estradiol, insulin-like growth factor-1, insulin, cholesterol and non-esterified fatty acids. Estrus was synchronized a second time, and liver and endometrial biopsies were collected on Day 7 of the estrous cycle. Gene expression was evaluated for a number of genes involved in prostaglandin synthesis (endometrium) and fatty acid uptake and utilization (liver). Fat supplementation had little effect on follicle development. Cows receiving supplementary n-3 fatty acids had lesser plasma progesterone (P4) and smaller corpora lutea than cows receiving the CLA or Control supplements. Effects of fat supplementation on the endometrial expression of genes involved in PG synthesis were minor. Hepatic expression of SREBF1, ASCL1 and FABP1 was reduced by FO supplementation. Reduced plasma P4 in n-3 supplemented cows may lead to a suboptimal uterine environment for embryo development and hence reduced fertility compared to cows receiving the control or CLA supplements.

Nutritional factors that regulate ovulation of the dominant follicle during the first follicular wave postpartum in high-producing dairy cows.

During recent decades, milk production per cow has increased drastically due to improved management, nutrition, and genetic selection; however, the reproductive performance of high-producing dairy cows has been declining. One of the factors responsible for this low reproductive performance is negative energy balance (NEB). NEB affects the onset of first ovulation in early postpartum cows. It is generally accepted that early first ovulation positively relates to the resumption of normal ovarian function, first service, and conception rate in dairy cows. Hence, delayed first ovulation has a negative impact on subsequent fertility. The metabolic condition of cows in NEB shifts to catabolic metabolism, which in turn causes increased plasma growth hormone and non-esterified fatty acid concentrations and decreased plasma insulin-like growth factor-1, insulin, and glucose concentrations. On the other hand, plasma ß-carotene concentrations decrease throughout the dry period and reach their nadir in about the first week postpartum, and this change reflects energy balance during the peripartum period. ß-Carotene plays a role independently of vitamin A in the reproductive performance of dairy cows, and the positive relationship between supplemental ß-carotene and reproductive function has been demonstrated in many studies during the past decades. However, ß-carotene content in corn silage, which is a popular main feed in high-producing dairy cows, is very low. This review describes nutritional factors related to ovulation during the first follicular wave postpartum in dairy cows.

Neuroendocrine control of ovulation.

Modern methods of diagnosis have made the distinction between hypothalamic failure and ovarian failure routine. Failure of the orderly progression of hypothalamic gonadotrophin-releasing hormone (GnRH) → pituitary gonadotrophins → ovarian steroids and inhibin → hypothalamus/pituitary results in anovulation/amenorrhea. The hypothalamic connections that regulate the pattern and amplitude of GnRH pulses are plastic and respond to external/psychological conditions and internal/metabolic factors that may affect the hypothalamic substrate on which estrogen levels can act. We trace the neuroendocrine regulation of the ovarian cycle, concentrating on hypothalamic connections that underlie negative and positive feedback control of GnRH and the complementary role of the adenohypophysis. The main hormone regulating this "central axis" and the development of the endometrium is estradiol which is exported from the developing ovarian follicles and thereby closes the feedback loop with follicle development. Progesterone and inhibin are also involved. Neuroendocrine responses to internal and external factors can cause anovulation and amenorrhea. Generally, these are accompanied by abnormal negative feedback between estradiol and the gonadotrophins; coexistence of low estradiol and luteinizing hormone/follicle-stimulating hormone. There are three main causes: (1) genetic diseases that interfere with the migration of GnRH cells into the brain or result in misfolding of GnRH; (2) input from the brain that interrupts normal feedback (e.g. stress and weight loss amenorrhea); and (3) the effect of agents which alter central neurotransmission and hypothalamic function (e.g. elevated prolactin and psychotropic medications). All types of hypothalamic insufficiency result in insufficient stimulation of the ovaries. In addition to amenorrhea, this central alteration also results in other complications (downstream disease) that make hypothalamic amenorrhea of greater consequence than simply reproductive failure. Thus, there may be more at stake in the diagnosis and treatment of hypothalamic failure than brings the patient to her caregiver.

CNS-specific ablation of steroidogenic factor 1 results in impaired female reproductive function.

The ventromedial hypothalamic nucleus (VMH) regulates a variety of homeostatic processes including female sexual behavior and reproduction. In the current study, we assessed the roles of steroidogenic factor 1 (SF-1) on reproductive function in the VMH using central nervous system-specific SF-1 knockout (SF-1 KO(nCre;F/-)) mice. Here we show that SF-1 KO(nCre;F/-) females exhibited marked impairment in female reproduction. Although male mice appeared to be normal in all aspects studied, including sexual behavior, SF-1 KO(nCre;F/-) females showed infertility or subfertility. Although adult SF-1 KO(nCre;F/-) females showed decreased or lacked corpora lutea, exogenous administration of gonadotropins induced the formation of multiple corpora lutea and induced normal ovulation, demonstrating that the ovaries are functionally intact. In addition, SF-1 KO(nCre;F/-) females stimulated with a synthetic GnRH agonist after priming exhibited markedly reduced LH secretion compared with wild-type littermates, arguing that disorganization in and around the VMH caused by SF-1 ablation interferes with the GnRH priming process or gonadotrope LH capacity. Furthermore, the SF-1 KO(nCre;F/-) females primed with estrogen benzoate and progesterone failed to induce steroid receptors around the VMH, consistent with impaired lordosis behavior in the SF-1 KO(nCre;F/-) females. Collectively, our results highlight that SF-1 in the VMH plays crucial roles in regulation of female reproductive function, presumably by organizing a precise neuronal connection and communication in and around the VMH.

Enhancement of ovulatory follicle development in maiden sheep by short-term supplementation with steam-flaked corn.

The effect of a short-term nutritional supplementation with steam-flaked corn on metabolism and folliculogenesis was evaluated in 14 maiden sheep. Oestrus was synchronized with two prostaglandin F(2alpha) doses given 10 days apart. From day 11 to 15 of the oestrous cycle induced with prostaglandins, half of the ewes (group 2M) were supplemented with steam-flaked corn, double the daily maintenance ration of the control sheep (group 1M). Body weight and condition remained unaffected, but the energetic supply increased plasma concentrations of glucose (3.6 +/- 0.1 vs 4.3 +/- 0.1 mmol/l, p < 0.0001) for the first 4 days and 3-hydroxybutyrate (0.323 +/- 0.58 vs 0.582 +/- 0.04 mmol/l, p < 0.005) from day 2 to 4. The profile of insulin secretion was also affected by the treatment, increasing in group 2M to reach significant differences on days 13 and 14 (p < 0.05). From similar values at the start of the food supply, the treatment induced a higher follicular development in group 2M (1.1 +/- 1.2 vs 7.4 +/- 1.06 total follicles in day 15, p < 0.05), as evidenced by the lineal increase in the number of larger follicles (>4 mm, p < 0.005). Then, the number of follicles >4 mm in size in 2M was around 60% higher on day 16 (7.86 +/- 0.45 vs 4.86 +/- 0.63, p < 0.005). Thereafter, the mean number of corpora lutea per ewe was around 30% higher in group 2M (1.43 +/- 0.2 vs 1.10 +/- 0.1, although differences were not found to be statistically significant). These data suggest that the use of diets containing high starch sources, like the steam-flaked corn, increases folliculogenesis and ovulation rate in sheep and can be applied in short-term feeding practices.

Atrazine and reproductive function: mode and mechanism of action studies.

Atrazine, a chlorotriazine herbicide, is used to control annual grasses and broadleaf weeds. In this review, we summarize our laboratory's work evaluating the neuroendocrine toxicity of atrazine (and related chlorotriazines) from an historic perspective. We provide the rationale for our work as we have endeavored to determine: 1) the underlying reproductive changes leading to the development of mammary gland tumors in the atrazine-exposed female rat; 2) the cascade of physiological events that are responsible for these changes (i.e., the mode of action for mammary tumors); 3) the potential cellular mechanisms involving adverse effects of atrazine; and 4) the range of reproductive alterations associated with this pesticide.

Changes in the level of endogenous leptin, FSH, 17beta-oestradiol and metabolites during lupin-induced increase in ovulation rate in ewes.

The aim of the study was to determine the changes in the plasma concentration of leptin during lupin feeding-induced increase in the ovulation rate (OR) in ewes. Additionally, alterations in the plasma level of glycogenic amino acids and glucose (as the factors influencing leptin secretion) and the levels of follicle-stimulating hormone (FSH) and 17beta-oestradiol (E-2) (as the hormones regulated by leptin and engaged in recruitment, selection and development of ovulatory follicles) were analysed. Ninety-six female Polish Lowland Sheep were used. All ewes were cyclic and synchronized with PGF2alpha. The ewes were divided into two groups: control (n = 48), fed only with hay, and experimental (n = 48), received additionally lupin (Lupinus angustifolius) grain as a high-protein and a high-energy supplement. They were given lupin from the second to 13th day of the oestrous cycle at increasing doses (150-750 g/day per ewe). On the 11th day of cycle blood samples for analysis of hormones, amino acids and total glucose concentration, were collected from the jugular vein. OR was determined by laparoscopy of ovaries on the sixth day of the following oestrous cycle. Mean OR of ewes supplemented with lupin grain (1.687 +/- 0.463) was 30.67% higher than that of control (1.291 +/- 0.454). In spite of the unchanged body mass, a significant increase (P < or = 0.05) in mean concentration of plasma leptin in the experimental ewes [2.17 +/- 0.15 ng/ml human equivalent (HE)] was found in comparison with control (1.42 +/- 0.12 ng/ml HE). A significantly (P < or = 0.05) higher plasma FSH level in the ewes fed lupin (105.21 +/- 5.87 ng/ml) compared with those fed hay (67.88 +/- 6.03 ng/ml) was also found. However, plasma level of E-2 decreased after lupin feeding. Moreover, in the ewes fed lupin the plasma concentrations of glucose and nine glycogenic amino acids (Gly, Ala, Val, Met, Leu, Ile, Tyr, Phe and Arg) were increased. It can be concluded that lupin feeding exerts the stimulatory effect on the OR in Polish Lowland Sheep. The increase in OR is connected with significantly higher plasma leptin level and coincident with rise in FSH, glycogenic amino acids and glucose concentration. In contrast, the level of plasma E-2 was significantly decreased in lupin-fed ewes.

GnRH antagonist inhibition of luteinizing hormone secretion and ovulation in the pig.

A GnRH antagonist, Ac-D-p-Cl-Phe1,2, D-Trp3, D-Arg6, D-Ala10 GnRHb (Organon), was utilized to determine the effective dosage and duration to inhibit LH secretion in the pig. In a preliminary trial, barrows received either 10, 50, or 250 micrograms/kg BW of the GnRH antagonist. Secretion of LH was inhibited within 30 min for a duration of 12 h with the 100 micrograms/kg dose but persisted for greater than 48 h with the 250 micrograms/kg treatment. A second study determined effectiveness of the antagonist for inhibiting ovulation in cyclic gilts. At first detection of standing estrus, cyclic gilts were treated with either saline (control), 100, or 200 micrograms/kg BW of the GnRH antagonist (GnRH1). A second group of GnRH antagonist gilts received 200 micrograms/kg BW of the GnRH antagonist approximately 8 h prior to standing estrus (GnRH2). The GnRH1-treatment failed to inhibit or delay ovulation. Ovulation was inhibited and estrous cycles lengthened in GnRH2-treated gilts. These preliminary results suggest that ovulation in the gilt can be inhibited if the GnRH antagonist is administered prior to the LH surge.

N-acetyl-beta-D-glucosaminidase as a marker of renal damage in hens.

Urinary N-acetyl-beta-D-glucosaminidase (NAG) is an early physiological indicator of renal damage in several mammalian species. A study was conducted to confirm occurrence of NAG in hen urine, to establish baseline urinary NAG in laying hens, and to assess the feasibility of using the enzyme as a marker of renal damage in hens. Hy-Line hens were used in a completely randomized block design in the first part of the study. Urine was collected at 4 to 6, 6 to 10, 10 to 14, and 14 to 18 h, and serum at 4, 6, 10, and 14 h postoviposition, and assayed by spectrophotometry for NAG. Kidney tissue from additional hens was assayed histochemically for NAG. Serum NAG (range: 0.11 to 0.14 mU/mg protein) was found to be several orders of magnitude lower than urine NAG (6.44 to 12.27 mU/mg protein). Urine NAG increased from 4 to 6 h through 14 to 18 h, indicating that time of collection is critical in order to utilize the enzyme as a valid marker for laying hens. A preliminary study with five hens indicated that 10 d of treatment with liquid cholecalciferol (D3) supplement (three times the recommended level) were not enough to detect renal damage on the basis of significant changes in urine (NAG, but elevated urine NAG was detected at 40 d of D3-supplementation. Overall the results indicate that NAG in urine of laying hens is a potentially useful diagnostic marker of renal damage.

Comparison of two multiple blood sampling regimens using an indwelling vascular access device for investigations of the hen's ovulatory cycle and calcium metabolism.

The objectives of the present study were to compare the effects of two regimens of serial blood sampling on the concentrations of hormones and ions during the ovulatory cycle of the domestic hen, and to examine the effectiveness of an indwelling vascular access device for repeated collection of blood samples. Single Comb White Leghorn hens were bled every 2 h over a period of 24 to 26 h, either from one oviposition to the next oviposition (OVIP-OVIP), or from 10 h prior to ovulation until the same time 24 h later (AFTN-AFTN). Whole blood was analyzed for ionized calcium concentration. Plasma was analyzed for total calcium, inorganic phosphorus, 1,25-dihydroxycholecalciferol, estradiol-17beta, and progesterone concentrations. The OVIP-OVIP regimen, using oviposition as a reference point, provided more accurate measurements of ionized calcium, inorganic phosphorus, and estradiol-17beta than did the AFTN-AFTN regimen. Either bleeding regimen was suitable for observing the patterns of 1,25-dihydroxycholecalciferol and progesterone concentrations. The decrease in bound calcium concentration observed with both regimens appeared to be an artifact of repeated blood sampling. The chance of a bird laying an egg following her second oviposition was lower following the OVIP-OVIP regimen than the AFTN-AFTN regimen. The vascular access device was a helpful tool in procuring multiple blood samples for measurement of ions and hormones during the ovulatory cycle of the domestic fowl.