Aspects of energetic substrate metabolism of in vitro and in vivo bovine embryos

Although the metabolism of early bovine embryos has not been fully elucidated, several publications have addressed this important issue to improve culture conditions for cattle reproductive biotechnologies, with the ultimate goal of producing in vitro embryos similar in quality to those developing in vivo. Here, we review general aspects of bovine embryo metabolism in vitro and in vivo, and discuss the use of metabolic analysis of embryos produced in vitro to assess viability and predict a viable pregnancy after transference to the female tract.

Culture of bovine ovarian follicle wall sections maintained the highly estrogenic profile under basal and chemically defined conditions

Follicle cultures reproduce in vitro the functional features observed in vivo. In a search for an ideal model, we cultured bovine antral follicle wall sections (FWS) in a serum-free defined medium (DM) known to induce 17β-estradiol (E2) production, and in a nondefined medium (NDM) containing serum. Follicles were sectioned and cultured in NDM or DM for 24 or 48 h. Morphological features were determined by light microscopy. Gene expression of steroidogenic enzymes and follicle-stimulating hormone (FSH) receptor were determined by RT-PCR; progesterone (P4) and E2 concentrations in the media were measured by radioimmunoassay. DM, but not NDM, maintained an FWS morphology in vitro that was similar to fresh tissue. DM also induced an increase in the expression of all steroidogenic enzymes, except FSH receptor, but NDM did not. In both DM and NDM, there was a gradual increase in P4 throughout the culture period; however, P4 concentration was significantly higher in NDM. In both media, E2 concentration was increased at 24 h, followed by a decrease at 48 h. The E2:P4 ratio was higher in DM than in NDM. These results suggest that DM maintains morphological structure, upregulates the expression of steroidogenic enzyme genes, and maintains steroid production with a high E2:P4 ratio in FWS cultures.

Prolactin and cortisol levels in women with endometriosis

Endometriosis is a progressive estrogen-dependent disease affecting women during their reproductive years. The objective of the present study was to investigate whether endometriosis is associated with stress parameters. We determined cortisol and prolactin levels in serum, peritoneal and follicular fluid from infertile women with endometriosis and fertile women without the disease. The extent of the disease was staged according to the revised American Fertility Society classification (1997). Serum and peritoneal fluid were collected from 49 women aged 19 to 39 years undergoing laparoscopy. Eighteen women had stage I-II endometriosis and 10 had stage III-IV. Controls were 21 women undergoing laparoscopy for tubal sterilization. Follicular fluid was obtained from 39 women aged 25-39 years undergoing in vitro fertilization (21 infertile women with endometriosis and 18 infertile women without endometriosis). Serum prolactin levels were significantly higher in infertile women with stage III-IV endometriosis (28.9 ± 2.1 ng/mL) than in healthy controls (13.2 ± 2.1 ng/mL). Serum cortisol levels were significantly higher in infertile women with stage III-IV endometriosis (20.1 ± 1.3 ng/mL) than in controls (10.5 ± 1.4 ng/mL). Cortisol and prolactin levels in follicular fluid and peritoneal fluid did not differ significantly between groups. The high levels of cortisol and prolactin in the serum from women with endometriosis might contribute to the subfertility frequently associated with the disease. Moreover, since higher levels of cortisol and prolactin are often associated with stress, it is probable that stress might contribute to the development of endometriosis and its progression to advanced stages of the disease.

Testosterona e gonadotrofina coriônica humana estimulam a esteroidogênese em células da granulosa de folículo pré-ovulatório de égua? / Do testosterone and human chorionic gonadotropin stimulate steroidogenesis in granulosa cells of preovulatory follicle in mare?

Avaliou-se o papel da gonadotrofina coriônica humana (hCG) e da testosterona na produção de progesterona (P4) e 17ß-estradiol (E2) pelas células da granulosa cultivadas in vitro de folículo antral de égua. Os tratamentos usados foram: 1- controle (nenhum hormônio adicionado), 2- 1UI hCG (0,3µg/ml) e 3- 10UI hCG (3,0µg/ml). O tratamento com hCG foi realizado na presença ou não de testosterona (144ng/ml). O meio foi coletado e substituído com 0,25, 3, 6, 12, 24 e 144h de cultivo. As concentrações de P4 e E2 foram mensuradas por radioimunoensaio. Não se observou diferença entre os tratamentos 1 e 3 quanto à produção de P4 e E2; o tratamento 1 resultou em aumento da concentração de progesterona após 24h de cultura (P<0,01), mas somente em presença de testosterona. A concentração de estradiol aumentou em presença de testosterona, alcançando concentração máxima com 6h de cultura (P<0,01), e diminuiu gradativamente, até atingir a concentração observada com 0,25h de cultura. A adição de hCG não influenciou a síntese do estradiol. A testosterona desempenhou importante efeito estimulador na síntese/secreção doe E2 pelas células da granulosa e modulou a ação do hormônio luteinizante na diferenciação e luteinização das células da granulosa de folículo antral presumidamente pré-ovulatório de égua in vitro.

Influência da alimentaçäo no teste de absorçäo e tolerância à glicose em eqüinos submetidos a diferentes períodos de jejum / Influence of nutrition on the glucose absorption and tolerance test in horses submitted to different fasting periods

Quatro grupos de eqüinos da raça Brasileiro de Hipismo foram submetidos a jejuns de 24 e 48 horas com a finalidade de se estudar a capacidade de absorçäo do intestino delgado. Dois grupos foram alimentados unicamente com capim coast cross (Cynodon dactylon). Os outros dois grupos, além de pasto de coast cross, receberam suplementaçäo com gräos. Ao final dos períodos de jejum, os animais receberam 1g de glucose/kg de peso corporal, em soluçäo a 20 por cento, por sonda nasogástrica. Amostras de sangue foram colhidas imediatamente antes, 30, 60, 120, 180, 240, 300 e 360 minutos após a administraçäo de glicose, para determinaçäo da glicemia pelo método da ortotoluidina e da insulina, pelo uso do radioimunoensaio. Os animais que receberam alimento concentrado apresentaram maiores aumentos na glicemia e na insulinemia que aqueles mantidos apenas em regime de pasto. O período de jejum de 48 horas induziu concentraçöes mais elevadas de glicemia e de insulinemia que o jejum de 24 horas

Decreased spermatogenic and androgenic testicular functions in adult rats submitted to immobilization-induced stress from prepuberty

We investigated whether chronic stress applied from prepuberty to full sexual maturity interferes with spermatogenic and androgenic testicular functions. Male Wistar rats (40 days old) were immobilized 6 h a day for 60 days. Following immobilization, plasma concentrations of corticosterone and prolactin increased 135 per cent and 48 per cent, respectively, while plasma luteinizing hormone and testosterone presented a significant decrease of 29 per cent and 37 per cent, respectively. Plasma concentration of follicle-stimulating hormone was not altered in stressed rats. Chronic stress reduced the amount of mature spermatids in the testis by 16 per cent and the spermatozoon concentration in the cauda epididymidis by 32 per cent. A 17 per cent reduction in weight and a 42 per cent decrease in DNA content were observed in the seminal vesicle of immobilized rats but not in its fructose content. The growth and secretory activity of the ventral prostate were not altered by chronic stress.

Chemical sympathectomy blocks androgen biosynthesis during prepuberty

Twenty-one-day old male Wistar rats were injected subcutaneously with guanethidine (GUA) at doses of 5 and 10 mg kg-1 day-1 for 20 days. Animals were sacrificed by decapitation during the prepubertal (41 days of age) and early-pubertal (51 days of age) periods of sexual development. The tests were collected, frozen in liquid N2 and stored at -70oC until determination of testicular progesterone (P), androstenedione (A) and testosterone (T). Higher levels of P (2.18 +/- 0.24 ng/g, control = 1.24 +/- 0.16 ng/g) associated with decreased with decreased levels of androgens (A = 0.26 +/- 0.06 ng/g T = 2.05 +/- 0.19 ng/g; control = 1.86 +/- 0.76 ng/g and 8.48 +/- 1.16 ng/g, respectively) were observed in 10 mg GUA-treated rats of prebubertal age, while only P levels (3.12 +/- 0.51 ng/g, control = 1.73 +/- 0.27 ng/g) were incresead in rats of early pubertal...

Relationship between plasma 17-beta estradiol on the day of estrus and number of viable embryos in Bos indicus (Nellore) cows superovulated with FSH

Thirteen cows, Bos indicus, of the Nellore breed were superovulated with 22 mg of follicle stimulating hormone (FSH) administered by intramuscular route during four consecutive days (D10, D11, D12 and D13), starting on the 10th day of the estrous cycle (day 0 = estrus). Prostaglandin (PGF2 alpha, 1.0 mg, im) was administered on D12, 48 h after the first FSH injection, for the induction of estrus on D14, when artificial insemination was performed. Seven days later (D21 of the cycle), embryos were collected, and evaluated, and the ovarian response was estimated on the basis of number of corpora lutea determined by rectal palpation. Blood samples were obtained for the determination of plasma 17-beta estradiol on D10, D11, D12, D13, D14 and D21 and plasma progesterone on D14 by RIA. The donors were divided into two groups according to progesterone levels on D14, the day of the induced estrus (GI: P4 < or = 1.00 ng/ml, N = 5 and GII: P4 > 1.00 ng/ml, N = 8). A linear positive correlation was observed between plasma 17-beta estradiol concentration on the day of estrus and viable embryo number. We conclude that plasma 17-beta estradiol and progesterone concentrations on the day of estrus can be used to predict the viability of embryos recovered from Nellore cows superovulated with FSH.

Acute administration of human chorionic gonadotropin reverts some of the inhibitory effects of an LH-RH agonist on testicular steroidogenesis

An LH-RH agonist, des-Gly10, [DTrp6]-LH-RH ethylamide (LH-RH A), was administered chronically to adult male cats in order to determine its effect on the steroidogenesis of the testis during the stimulatory action of human chorionic gonadotropin (hCG). Measurement of plasma testosterone levels were combined with the histochemical analysis of some steps of the testicular steroidogenic pathway. Chronic daily treatment with LH-RH A(20 *g/kg) for 67 days inhibited the early testicular response to hCG during the initial 0.5,1 and 24 h, whereas the inhibitory egffect was not observed 48 and 72 h after hCG administration.The maximal responses to hCG were obtained both in LH-RH A-treated animals and in their control group 48 and 72 h after hCG adnministration. Under these conditions, LH-RH A-treated cats showed no alteration in 3ß-hydroxysteroid dehydrogenase (3ß-Host-D) activity, whereas a marked inhibition was observed in the activity of alcohol dehydrogenase (ADII) which reflects the activity of 20,22-desmolase and/or 17,20-desmolase.