Subacute exposure to Roundup® changes steroidogenesis and gene expression of the glutathione-glutaredoxin system in rat ovaries: Implications for ovarian toxicity of this glyphosate-based herbicide.

Studies have indicated that glyphosate induces endocrine disruption and may adversely affect the male reproductive system. However, evidence of its effects on ovarian function is poorly understood so far, making further studies necessary on the mechanisms of the glyphosate toxicity in the female reproductive system. The aim of this work was to evaluate the effect of a subacute exposure (28 days) to the glyphosate-based formulation Roundup® (1.05, 10.5 and 105 µg/kg b.w. of glyphosate) on steroidogenesis, oxidative stress, systems involved in cell redox control and histopathological parameters in rat ovaries. Hence we quantify plasma estradiol and progesterone by chemiluminescence; non-protein thiol levels, TBARS, superoxide dismutase and catalase activity by spectrophotometry; gene expression of steroidogenic enzymes and redox systems by real-time PCR; and ovarian follicles by optical microscopy. Our results demonstrated that oral exposure increased progesterone levels and the mRNA expression of 3ß-hydroxysteroid dehydrogenase. Histopathological analysis revealed a decrease in the number of primary follicles and an increase in the number of corpus luteum in rats exposed to Roundup®. An imbalance of the oxidative status was also evidenced by decreasing the catalase activity at all groups exposed to the herbicide. Increased lipid peroxidation and gene expression of glutarredoxin and decreased of glutathione reductase were also observed. Our results indicate that Roundup® causes endocrine disruption of hormones related to female fertility and reproduction and changes the oxidative status by altering antioxidant activity, inducing lipid peroxidation, as well as changing the gene expression of the glutathione-glutarredoxin system in rat ovaries.

Regulation and function of leptin during ovarian follicular development in cows.

Although it is well documented that leptin signals the body nutritional status to the brain, mechanisms of leptin regulation at the ovary are not well understood. This study was conducted to determine whether there was leptin and the receptor for leptin (LEPR) in cattle ovarian follicles and to investigate potential actions of leptin on follicular growth in vivo and on regulation of granulosa cell functions in vitro. There was leptin and LEPR in granulosa and theca cells of dominant and subordinate follicles, with greater immunostaining for leptin in granulosa cells of subordinate follicles. There was a lesser relative abundance of leptin receptor gene-related protein (LEPROT) and of the adiponectin receptors 1 (ADIPOR1) and 2 (ADIPOR2) mRNA transcripts in granulosa cells of subordinate than dominant follicles (P < 0.05). Intrafollicular injection of either 100 or 1000 ng/mL leptin did not affect the diameter and the growth of dominant follicles (P> 0.05). Supplementation of in vitro culture medium with different leptin concentations did not affect (P > 0.05) the relative abundance of hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (HSD3B1), cytochrome P450 family 11 subfamily A member 1 (CYP11A1), signal transducer and activator of transcription 3 (STAT3) and X-linked inhibitor of apoptosis protein (XIAP) mRNA transcripts in granulosa cells. These findings indicate that leptin and LEPR are present in the follicular cells of cattle ovaries, but leptin apparently does not have essential functions in steroidogenesis and growth of dominant follicles.

Effects of the mycotoxin metabolite de-epoxy-deoxynivalenol (DOM-1) on embryo development and sperm motility in cattle.

Contamination of animal feed with Fusarium spp results in accumulation of mycotoxins including deoxynivalenol. In animals, deoxynivalenol is metabolized to de-epoxy deoxynivalenol (DOM-1), which is generally considered to be a non-toxic metabolite; however, recent studies demonstrated that DOM-1 can reduce steroid production and induce apoptosis in the bovine ovary. The objectives of this study were to assess the effects of DOM-1 on applied aspects of reproductive function in cattle, specifically sperm function and embryo development in vitro and follicle growth and superovulatory responses in vivo. The effect of naturally contaminated feed on superovulatory responses was assessed; a dose of 6 ppm deoxynivalenol increased blood DOM-1 concentrations to 20 ng/ml, but this did not alter the number of viable embryos recovered on day 7. However, intrafollicular injection of DOM-1 (100 ng/ml) directly into the growing dominant follicle resulted in cessation of follicular growth over the subsequent 3 days. Treatment with DOM-1 reduced motility of bull spermatozoa over a 10-h period in vitro. Addition of DOM-1 to oocytes in vitro during IVM did not alter rates of cumulus expansion and nuclear maturation, but treatment during IVF reduced the rate of blastocyst formation. These data illustrate that DOM-1 is more biologically active than previously thought and negatively impacted reproductive outcomes in cattle.

Evaluation of interferon-stimulated gene 15 (ISG15) expression in blood neutrophils in beef cattle poisoned by Senecio spp / Avaliação da expressão do gene estimulado por interferon 15 (ISG15) em neutrófilos sanguíneos em bovinos de corte intoxicados por Senecio spp

This study aimed to assess liver damage and interferon-stimulated gene 15 (ISG15) blood expression as a consequence of embryonic signaling on maternal recognition of pregnancy in beef cattle presenting natural ingestion of Senecio spp. Epidemiological aspects, as the presence of the plant, associated to gamma glutamyl transferase (GGT) activity can be used as Senecio spp. poisoning diagnosis. Maternal recognition of pregnancy period occurs when the embryo secretes interferon tau (IFNT) to signal its presence to the mother and eventually extend corpus luteum (CL) lifespan. In our study, liver damage was determined by concentration serum GGT, cytological and histopathological examinations. Reproductive status was evaluated by concentration of progesterone, CL diameter and ISG15 mRNA expression on Day 19 following fixed-time artificial insemination (FTAI). Cows were categorized into two groups based on concentration of GGT: Group 1 (GGT<30U/L) and 2 (GGT>31U/L). No difference on body condition scores was observed. All the cows presented liver damage based on cytology and histopathological exams. Cows from the Group 1 had higher pregnancy rate, presenting larger CL diameter and greater concentration of progesterone. Interestingly, ISG15 mRNA expression had no difference between Groups 1 and 2, even presenting difference in pregnancy status. These findings suggest embryonic loss beyond Day 19. It suggests late embryonic mortality may be associated to liver insufficiency. In conclusion, liver injury and/or concentration of GGT does not alter ISG15 expression on blood neutrophils, however cows presenting lower concentration of GGT (<30U/L) had increased pregnancy status. Therefore, the concentration of GGT allow us to screen liver status and foresee a successful pregnancy in beef cattle.(AU)

O objetivo deste estudo foi avaliar a lesão hepática e a expressão sanguínea do gene estimulado por interferon 15 (ISG15) durante a sinalização embrionária, no reconhecimento materno da gestação, em bovinos de corte apresentando ingestão natural de Senecio spp. Fatores epidemiológicos, como a presença da planta, associados à atividade da gama glutamil transferase (GGT) podem ser utilizados como diagnóstico da intoxicação por Senecio spp. O reconhecimento materno da gestação ocorre quando o embrião secreta interferon tau (IFNT) para sinalizar sua presença à mãe. Em nosso estudo, a lesão hepática foi determinada pela concentração sérica de GGT, pelos exames citológicos e histopatológicos. O estado reprodutivo foi avaliado pela concentração de progesterona, diâmetro de corpo lúteo (CL) e expressão de mRNA ISG15 no Dia 19 após a inseminação artificial em tempo fixo (IATF). As vacas foram separadas em dois grupos com base na concentração de GGT sanguíneo: Grupo 1 (GGT<30U/L) e Grupo 2 (GGT>31U/L). Não foi observada nenhuma diferença no escore de condição corporal entre os grupos. Na citologia e nos exames histopatológicos todas as vacas apresentaram lesão hepática. As vacas do Grupo 1 apresentaram maior taxa de prenhez, maior diâmetro do CL e maior concentração de progesterona. Diferente do esperado, a expressão do mRNA ISG15 não foi diferente entre os Grupos 1 e 2, mesmo apresentando diferença na taxa de prenhez. Esses achados sugerem perda embrionária após o Ddia 19. Isso demonstra que a mortalidade embrionária tardia pode estar associada à insuficiência hepática. Dessa forma, conclui-se que a lesão hepática e/ou concentração de GGT não altera a expressão de ISG15 nos neutrófilos sanguíneos, porém vacas com menor concentração de GGT (<30U/L) apresentaram maiores taxas de prenhez. Assim, a concentração de GGT nos permite avaliar a saúde hepática e prever uma gestação bem-sucedida em bovinos de corte.(AU)

Evaluation of interferon-stimulated gene 15 (ISG15) expression in blood neutrophils in beef cattle poisoned by Senecio spp

ABSTRACT: This study aimed to assess liver damage and interferon-stimulated gene 15 (ISG15) blood expression as a consequence of embryonic signaling on maternal recognition of pregnancy in beef cattle presenting natural ingestion of Senecio spp. Epidemiological aspects, as the presence of the plant, associated to gamma glutamyl transferase (GGT) activity can be used as Senecio spp. poisoning diagnosis. Maternal recognition of pregnancy period occurs when the embryo secretes interferon tau (IFNT) to signal its presence to the mother and eventually extend corpus luteum (CL) lifespan. In our study, liver damage was determined by concentration serum GGT, cytological and histopathological examinations. Reproductive status was evaluated by concentration of progesterone, CL diameter and ISG15 mRNA expression on Day 19 following fixed-time artificial insemination (FTAI). Cows were categorized into two groups based on concentration of GGT: Group 1 (GGT 30U/L) and 2 (GGT>31U/L). No difference on body condition scores was observed. All the cows presented liver damage based on cytology and histopathological exams. Cows from the Group 1 had higher pregnancy rate, presenting larger CL diameter and greater concentration of progesterone. Interestingly, ISG15 mRNA expression had no difference between Groups 1 and 2, even presenting difference in pregnancy status. These findings suggest embryonic loss beyond Day 19. It suggests late embryonic mortality may be associated to liver insufficiency. In conclusion, liver injury and/or concentration of GGT does not alter ISG15 expression on blood neutrophils, however cows presenting lower concentration of GGT ( 30U/L) had increased pregnancy status. Therefore, the concentration of GGT allow us to screen liver status and foresee a successful pregnancy in beef cattle.

RESUMO: O objetivo deste estudo foi avaliar a lesão hepática e a expressão sanguínea do gene estimulado por interferon 15 (ISG15) durante a sinalização embrionária, no reconhecimento materno da gestação, em bovinos de corte apresentando ingestão natural de Senecio spp. Fatores epidemiológicos, como a presença da planta, associados à atividade da gama glutamil transferase (GGT) podem ser utilizados como diagnóstico da intoxicação por Senecio spp. O reconhecimento materno da gestação ocorre quando o embrião secreta interferon tau (IFNT) para sinalizar sua presença à mãe. Em nosso estudo, a lesão hepática foi determinada pela concentração sérica de GGT, pelos exames citológicos e histopatológicos. O estado reprodutivo foi avaliado pela concentração de progesterona, diâmetro de corpo lúteo (CL) e expressão de mRNA ISG15 no Dia 19 após a inseminação artificial em tempo fixo (IATF). As vacas foram separadas em dois grupos com base na concentração de GGT sanguíneo: Grupo 1 (GGT 30U/L) e Grupo 2 (GGT>31U/L). Não foi observada nenhuma diferença no escore de condição corporal entre os grupos. Na citologia e nos exames histopatológicos todas as vacas apresentaram lesão hepática. As vacas do Grupo 1 apresentaram maior taxa de prenhez, maior diâmetro do CL e maior concentração de progesterona. Diferente do esperado, a expressão do mRNA ISG15 não foi diferente entre os Grupos 1 e 2, mesmo apresentando diferença na taxa de prenhez. Esses achados sugerem perda embrionária após o Ddia 19. Isso demonstra que a mortalidade embrionária tardia pode estar associada à insuficiência hepática. Dessa forma, conclui-se que a lesão hepática e/ou concentração de GGT não altera a expressão de ISG15 nos neutrófilos sanguíneos, porém vacas com menor concentração de GGT ( 30U/L) apresentaram maiores taxas de prenhez. Assim, a concentração de GGT nos permite avaliar a saúde hepática e prever uma gestação bem-sucedida em bovinos de corte.

The endogenous hydrogen sulfide generating system regulates ovulation.

The generation of free-radicals such as nitric oxide has been implicated in the regulation of ovarian function, including ovulation. Tissues that generate nitric oxide typically generate another free-radical gas, hydrogen sulfide (H2S), although little is known about the role of H2S in ovarian function. The hypothesis of this study was that H2S regulates ovulation. Treatment with luteinizing hormone (LH) increased the levels of mRNA and protein of the H2S generating enzyme cystathionine γ-lyase (CTH) in granulosa cells of mice and humans in vivo and in vitro. Pharmacological inhibition of H2S generating enzymes reduced the number of follicles ovulating in mice in vivo and in vitro, and this inhibitory action was reversed by cotreatment with a H2S donor. Addition of a H2S donor to cultured mouse granulosa cells increased basal and LH-dependent abundance of mRNA encoding amphiregulin, betacellulin and tumor necrosis alpha induced protein 6, proteins important for cumulus expansion and follicle rupture. Inhibition of CTH activity reduced abundance of mRNA encoding matrix metalloproteinase-2 and -9 and tissue-type plasminogen activator, and cotreatment with the H2S donor increased the levels of these mRNA above those stimulated by LH alone. We conclude that the H2S generating system plays an important role in the propagation of the preovulatory cascade and rupture of the follicle at ovulation.

The role of fibroblast growth factor-18 in follicular atresia in cattle.

Although the various members of the fibroblast growth factor (FGF) family are generally mitotic, one member, FGF18, has been shown to increase the rate of apoptosis of ovarian granulosa cells. In the present study, we first determined whether granulosa cells express FGF18 and we then explored the mechanism through which FGF18 increases apoptosis in vitro. Under culture conditions that favored estradiol secretion and CYP19A1 expression, granulosa FGF18 mRNA levels were barely detectable; however, withdrawing gonadotropic support (follicle-stimulating hormone or insulin-like growth factor 1) reduced levels of CYP19A1 mRNA and increased abundance of mRNA encoding the death ligand FASLG and FGF18. Addition of FGF18, but not FGF2, FGF10, or EGF, increased the proportion of apoptotic cells and frequency of caspase 3 activation, and these effects were abrogated by coculture with estradiol. Addition of FGF18 decreased abundance of mRNA encoding the antiapoptotic proteins GADD45B and MDM2, and increased that encoding the proapoptotic protein BBC3; these effects were reversed by coculture with estradiol. The physiological relevance of FGF18 was determined using an in vivo model: injection of FGF18 directly into growing bovine dominant follicles caused cessation of follicle growth by 24 h after injection. Collectively, these data demonstrate that FGF18 is proapoptotic in vivo and may act through a mechanism involving the BBC3-MDM2 pathway.

Role of angiotensin II in the periovulatory epidermal growth factor-like cascade in bovine granulosa cells in vitro.

Angiotensin II (AGT-2) induces follicular prostaglandin release in a number of species and ovulation in rabbits. Conversely, AGT-2 antagonists block ovulation in cattle. To determine the mechanism of action of AGT-2, we used a bovine granulosa cell model in which luteinizing hormone (LH) increased the expression of genes essential for ovulation in a time- and dose-dependent manner. The addition of AGT-2 to LH-stimulated cells significantly increased abundance of prostaglandin-endoperoxide synthase 2 (PTGS2) mRNA and protein, whereas AGT-2 alone had no effect. Upstream of PTGS2, AGT-2 increased abundance of mRNA encoding the epidermal growth factor-like ligands amphiregulin (AREG) and epiregulin (EREG) at 6 h posttreatment and abundance of a disintegrin and metalloprotease 17 (ADAM17), a sheddase, within 3 h of treatment. Inhibiting sheddase activity abolished the stimulatory effect of AGT-2 on AREG, EREG, and PTGS2 mRNA. The addition of selective AGT-2 antagonists to cells stimulated with LH plus AGT-2 demonstrated that AGT-2 did not act through the type 1 receptor and did not increase mitogen-activated protein kinase 3/1 phosphorylation. Combined with previous data from studies in vitro, we conclude that AGT-2 is an essential cofactor for LH in the early increase of ADAM expression/activity that induces the cascade of events leading to ovulation.

Effect of medroxy-progesterone acetate on follicular growth and endometrial cycloxygenase-2 (COX-2) expression during the bovine estrous cycle / Efeito do acetato de medroxi-progesterona sobre o crescimento filicular e expressão endometrial de ciclooxigenase-2 (COX-2) durante o ciclo estral de bovinos

The objective of this study was to evaluate the effect of medroxy-progesterone acetate (MAP) with or without estradiol benzoate (EB) on follicular growth during the estrous cycle in cattle. In the first experiment, Hereford cows were synchronized with a synthetic analogue of PGF2 alpha and were treated with two different doses of MAP (250 or 500 mg) with or without EB for 7 days starting on day 8 of the estrous cycle. Follicular growth was inhibited (P<0.05) in all cows except controls and those receiving 250mg MAP without EB. Seventy-five percent of the animals (15/20) showed estrus on days 21 and 22 of the cycle rather than at MAP withdrawal, demonstrating that these treatments did not induce estrus. To determine whether the EB treatment altered endometrial sensitivity to oxytocin and thus the luteolytic cascade, multiparous pre-synchronized cows received 5 mg of EB followed 6 hours later with 50 IU of oxytocin (OT; n=9). Eight hours after EB injection, endometrial fragments were collected from the cows on days 4, 13 and 17 of the estrous cycle and COX-2 gene expression was measured by PCR. EB increased COX-2 mRNA levels only on day 17 of the estrous cycle (P<0.05). In conclusion, MAP alone or associated with EB is able to suppress bovine follicular growth. However, EB in the presence of MAP is not efficient to induce luteolysis in cows when injected on day 8 of the estrous cycle.

Este estudo teve como objetivo avaliar o efeito do acetato de medroxi-progesterona (MAP) com ou sem benzoato de estradiol (BE) sobre o crescimento folicular durante o ciclo estral bovino. No primeiro experimento, vacas da raça Hereford foram sincronizadas com um análogo sintético de PGF2á e tratadas com duas doses diferentes de MAP (250 ou 500mg), com ou sem EB, durante 7 dias, iniciando-se no oitavo dia do ciclo estral. Observou-se uma inibição do crescimento folicular (P<0,05) em todas as vacas, exceto no grupo controle e no grupo que recebeu 250mg de MAP sem BE. Os 75 por cento dos animais não exibiu estro no momento da remoção do MAP, mas sim nos dias 21 e 22 do ciclo, demonstrando que os tratamentos não induziram cio. Para se determinar se o tratamento com BE alterou a sensibilidade endometrial à ocitocina e, assim, a cascata luteolítica, vacas multíparas pré-sincronizadas receberam 5mg de BE, seguidos, após 6 horas, de 50 UI de ocitocina (OT; n=9). Oito horas após a administração de BE, colheram-se fragmentos endometriais das vacas, nos dias 4, 13 e 17 do ciclo estral, mensurando-se a expressão gênica de COX-2 através de PCR. O BE aumentou os níveis de RNAm de COX-2 apenas no dia 17 do ciclo estral (P<0,05). Em conclusão, o MAP isolado ou associado a BE é capaz de suprimir o crescimento folicular bovino. Entretanto, o BE, na presença de MAP é ineficaz na indução da luteólise bovina, quando injetado no oitavo dia do ciclo estral.

Expression and function of fibroblast growth factor 18 in the ovarian follicle in cattle.

Fibroblast growth factors (FGF) are involved in paracrine signaling between cell types in the ovarian follicle. FGF8, for example, is secreted by oocytes and controls cumulus cell metabolism. The closely related FGF18 is also expressed in oocytes in mice. The objective of this study was to assess the potential role of FGF18 in follicle growth in a monovulatory species, the cow. Messenger RNA encoding FGF18 was detected primarily in theca cells, and in contrast to the mouse, FGF18 was not detected in bovine oocytes. Addition of FGF18 protein to granulosa cell cultures inhibited estradiol and progesterone secretion as well as the abundance of mRNA encoding steroidogenic enzymes and the follicle-stimulating hormone receptor. In vivo, onset of atresia of the subordinate follicle was associated with increased thecal FGF18 mRNA levels and FGF18 protein in follicular fluid. In vitro, FGF18 altered cell cycle progression as measured by flow cytometry, resulting in increased numbers of dead cells (sub-G1 peak) and decreased cells in S phase. This was accompanied by decreased levels of mRNA encoding the cell cycle checkpoint regulator GADD45B. Collectively, these data point to a unique role for this FGF in signaling from theca cells to granulosa cells and suggest that FGF18 influences the process of atresia in ovarian follicles.

Cell plating density alters the ratio of estrogenic to progestagenic enzyme gene expression in cultured granulosa cells.

OBJECTIVE: To determine if initial cell plating density alters steroidogenesis and the E(2):P ratio in granulosa cells in long-term serum-free culture. DESIGN: Experimental study. SETTING: Academic institution. ANIMAL(S): Cattle of slaughterhouse origin. INTERVENTION(S): Culture of granulosa cells in vitro at different cell plating density. MAIN OUTCOME MEASURE(S): Steroid secretion was measured by RIA, mRNA levels were measured by real-time polymerase chain reaction, and cell death was assessed by flow cytometry. RESULT(S): Low plating density favored E(2) secretion and mRNA encoding estrogenic enzymes, whereas higher density inhibited E(2) secretion and enhanced P secretion and levels of mRNA encoding progestagenic enzymes. Increasing plating density decreased the E(2):P ratio and cell health. CONCLUSION(S): Lower cell density favors an estrogenic granulosa cell phenotype, whereas higher density favors luteinization. Serum-free culture systems should be optimized with this in mind.

Regulation of MMP2 and MMP9 metalloproteinases by FSH and growth factors in bovine granulosa cells.

Matrix metalloproteinases (MMP) are key enzymes involved in tissue remodeling. Within the ovary, they are believed to play a major role in ovulation, and have been linked to follicle atresia. To gain insight into the regulation of MMPs, we measured the effect of hormones and growth factors on MMP2 and MMP9 mRNA levels in non-luteinizing granulosa cells in serum-free culture. FSH and IGF1 both stimulated estradiol secretion and inhibited MMP2 and MMP9 mRNA abundance. In contrast, EGF and FGF2 both inhibited estradiol secretion but had no effect on MMP expression. At physiological doses, none of these hormones altered the proportion of dead cells. Although we cannot link MMP expression with apoptosis, the specific down regulation by the gonadotropic hormones FSH and IGF1 in vitro suggests that excess MMP2 and MMP9 expression is neither required nor desired for follicle development.

Regulation of MMP2 and MMP9 metalloproteinases by FSH and growth factors in bovine granulosa cells

Matrix metalloproteinases (MMP) are key enzymes involved in tissue remodeling. Within the ovary, they are believed to play a major role in ovulation, and have been linked to follicle atresia. To gain insight into the regulation of MMPs, we measured the effect of hormones and growth factors on MMP2 and MMP9 mRNA levels in non-luteinizing granulosa cells in serum-free culture. FSH and IGF1 both stimulated estradiol secretion and inhibited MMP2 and MMP9 mRNA abundance. In contrast, EGF and FGF2 both inhibited estradiol secretion but had no effect on MMP expression. At physiological doses, none of these hormones altered the proportion of dead cells. Although we cannot link MMP expression with apoptosis, the specific down regulation by the gonadotropic hormones FSH and IGF1 in vitro suggests that excess MMP2 and MMP9 expression is neither required nor desired for follicle development.

Regulation of angiotensin type 2 receptor in bovine granulosa cells.

Angiotensin II (AngII) is best known for its role in blood pressure regulation, but it also has documented actions in the reproductive system. There are two AngII receptors, type 1 (AGTR1) and type 2 (AGTR2). AGTR2 mediates the noncardiovascular effects of AngII and is expressed in the granulosa cell layer in rodents and is associated with follicle atresia. In contrast, expression of AGTR2 is reported to occur only in theca cells in cattle. The objective of the present study was to determine whether AngII also plays a role in follicle atresia in cattle. RT-PCR demonstrated AGTR2 mRNA in both granulosa and theca cells of bovine follicles. The presence of AGTR2 protein was confirmed by immunofluorescence. Abundance of AGTR2 mRNA in granulosa cells was higher in healthy compared with atretic follicles, whereas in theca cells, it did not change. Granulosa cells were cultured in serum-free medium, and treatment with hormones that increase estradiol secretion (FSH, IGF-I, and bone morphogenetic protein-7) increased AGTR2 mRNA and protein levels, whereas fibroblast growth factors inhibited estradiol secretion and AGTR2 protein levels. The addition of AngII or an AGTR2-specific agonist to granulosa cells in culture did not affect estradiol secretion or cell proliferation but inhibited abundance of mRNA encoding serine protease inhibitor E2, a protein involved in tissue remodeling. Because estradiol secretion is a major marker of nonatretic granulosa cells, these data suggest that AngII is not associated with follicle atresia in cattle but may have other specific roles during follicle growth.

Regulation of serine protease inhibitor-E2 and plasminogen activator expression and secretion by follicle stimulating hormone and growth factors in non-luteinizing bovine granulosa cells in vitro.

During ovarian follicle growth, there is expansion of the basal lamina and changes in the follicular extracellular matrix (ECM) that are mediated in part by proteolytic enzyme cascades regulated by tissue-type plasminogen activator (tPA) and urokinase plasminogen activator (uPA). One PA inhibitor, serine protease inhibitor-E2 (SERPINE2) is expressed in granulosa but not theca cells, and expression changes with follicle development. In this study, we hypothesized that PA and SERPINE2 expression/secretion by granulosa cells are regulated by FSH and growth factors. SERPINE2 mRNA and protein levels, tPA gene expression and uPA secretion were stimulated by FSH. Insulin-like growth factor-I stimulated SERPINE2 secretion and uPA activity, and decreased secreted tPA activity and gene expression. Bone morphogenetic protein-7 increased SERPINE2 secretion and expression and tPA secretion. In contrast, fibroblast growth factor-2 inhibited tPA secretion and SERPINE2 secretion and expression. Epidermal growth factor inhibited SERPINE2 secretion and expression, but increased secreted tPA activity. Estradiol and SERPINE2 secretion were highly positively correlated, but estradiol did not alter SERPINE2 expression. These data demonstrate that SERPINE2 expression and protein secretion are regulated by FSH and growth factors in non-luteinizing bovine granulosa cells. As estradiol is a known marker of follicle health, and SERPINE2 is an anti-apoptotic factor, we propose that SERPINE2 is involved in the regulation of atresia in bovine follicles.