Early prenatal androgen exposure reduces testes size and sperm concentration in sheep without altering neuroendocrine differentiation and masculine sexual behavior.

Prenatal androgens are largely responsible for growth and differentiation of the genital tract and testis and for organization of the control mechanisms regulating male reproductive physiology and behavior. The aim of the present study was to evaluate the impact of inappropriate exposure to excess testosterone (T) during the first trimester of fetal development on the reproductive function, sexual behavior, and fertility potential of rams. We found that biweekly maternal T propionate (100 mg) treatment administered from Day 30-58 of gestation significantly decreased (P < 0.05) postpubertal scrotal circumference and sperm concentration. Prenatal T exposure did not alter ejaculate volume, sperm motility and morphology or testis morphology. There was, however, a trend for more T-exposed rams than controls to be classified as unsatisfactory potential breeders during breeding soundness examinations. Postnatal serum T concentrations were not affected by prenatal T exposure, nor was the expression of key testicular genes essential for spermatogenesis and steroidogenesis. Basal serum LH did not differ between treatment groups, nor did pituitary responsiveness to GnRH. T-exposed rams, like control males, exhibited vigorous libido and were sexually attracted to estrous females. In summary, these results suggest that exposure to exogenous T during the first trimester of gestation can negatively impact spermatogenesis and compromise the reproductive fitness of rams.

Effects of Long-Term Flutamide Treatment During Development on Sexual Behaviour and Hormone Responsiveness in Rams.

Testosterone exposure during midgestation differentiates neural circuits controlling sex-specific behaviours and patterns of gonadotrophin secretion in male sheep. Testosterone acts through androgen receptors (AR) and/or after aromatisation to oestradiol and binding to oestrogen receptors. The present study assessed the role of AR activation in male sexual differentiation. We compared rams that were exposed to the AR antagonist flutamide (Flu) throughout the critical period (i.e. days 30-90 of gestation) to control rams and ewes that received no prenatal treatments. The external genitalia of all Flu rams were phenotypically female. Testes were positioned s.c. in the inguinal region of the abdomen, exhibited seasonally impaired androgen secretion and were azospermic. Flu rams displayed male-typical precopulatory and mounting behaviours but could not intromit or ejaculate because they lacked a penis. Flu rams exhibited greater mounting behaviour than control rams and, similar to controls, showed sexual partner preferences for oestrous ewes. Neither control, nor Flu rams responded to oestradiol treatments with displays of female-typical receptive behaviour or LH surge responses, whereas all control ewes responded as expected. The ovine sexually dimorphic nucleus in Flu rams was intermediate in volume between control rams and ewes and significantly different from both. These results indicate that prenatal anti-androgen exposure is not able to block male sexual differentiation in sheep and suggest that compensatory mechanisms intervene to maintain sufficient androgen stimulation during development.

Sex differences in expression of oestrogen receptor α but not androgen receptor mRNAs in the foetal lamb brain.

Gonadal steroid hormones play important roles during critical periods of development to organise brain structures that control sexually dimorphic neuroendocrine responses and behaviours. Specific receptors for androgens and oestrogens must be expressed at appropriate times during development to mediate these processes. The present study was performed to test for sex differences in the relative expression of oestrogen receptor (ER)α and androgen receptor (AR) mRNA during the window of time in gestation that is critical for behavioural masculinisation and differentiation of the ovine sexually dimorphic nucleus (oSDN) in the sheep. In addition, we examined whether ERα and AR mRNA expression is localised within the nascent oSDN and could be involved in its development. Using the quantitative real-time polymerase chain reaction, we found that females expressed more ERα mRNA than males in medial preoptic area and medial basal hypothalamus during the mid-gestational critical period for brain sexual differentiation. No sex differences were found for AR mRNA in any tissue examined or for ERα in amygdala and frontal cortex. Using radioactive in situ hybridisation, we found that the distributions of ERα and AR mRNA overlapped with aromatase mRNA, which delineates the boundaries of the developing oSDN and identifies this nucleus as a target for both androgens and oestrogens. These data demonstrate that the transcriptional machinery for synthesising gonadal steroid receptors is functional in the foetal lamb brain during the critical period for sexual differentiation and suggest that possible mechanisms for establishing dimorphisms controlled by gonadal steroids may exist at the level of steroid hormone receptor expression.

Ontogeny of cytochrome p450 aromatase mRNA expression in the developing sheep brain.

The intraneuronal conversion of testosterone to oestradiol constitutes a critical step in the development and sexual differentiation of the brain of many short gestation mammalian species and has been inferred to play a similar role in long gestation sheep. This conversion is catalysed by cytochrome P450 aromatase (CYP19), which is expressed in specific brain structures during foetal development. The present study was undertaken to examine the specific neuroanatomical distribution and relative expression of aromatase mRNA in the developing sheep hypothalamus. The foetal sheep is a highly tractable model system for localising the region-specific expression of aromatase in the brain during prenatal development that can help predict regions where oestrogen acts to shape neural development. Our results, obtained using real time quantitative reverse transcriptase-polymerase chain reaction, revealed that aromatase mRNA was expressed throughout mid to late gestation in the foetal preoptic area and amygdala. In the preoptic area, aromatase expression declined with advancing gestation, whereas, it increased in the amygdala. No sex differences were observed in either brain area. We next investigated the anatomical distribution of aromatase using in situ hybridisation histochemistry and found that the pattern of mRNA expression was largely established by midgestation. High expression was observed in the medial preoptic nucleus, bed nucleus of the stria terminalis and corticomedial amygdala. We also observed substantial expression in the dorsal striatum. These results extend our understanding of the developmental expression of aromatase in the foetal sheep brain and lend support to the view that it plays an essential role in sexual differentiation and maturation of the neuroendocrine, motor and reward control systems.

The ovine sexually dimorphic nucleus, aromatase, and sexual partner preferences in sheep.

We are using the domestic ram as an experimental model to examine the role of aromatase in the development of sexual partner preferences. This interest has arisen because of the observation that as many as 8% of domestic rams are sexually attracted to other rams (male-oriented) in contrast to the majority of rams that are attracted to estrous ewes (female-oriented). Our findings demonstrate that aromatase expression is enriched in a cluster of neurons in the medial preoptic nucleus called the ovine sexually dimorphic nucleus (oSDN). The size of the oSDN is associated with a ram's sexual partner preference, such that the nucleus is 2-3 times larger in rams that are attracted to females (female-oriented) than in rams that are attracted to other rams (male-oriented). Moreover, the volume of the oSDN in male-oriented rams is similar to the volume in ewes. These volume differences are not influenced by adult concentrations of serum testosterone. Instead, we found that the oSDN is already present in late gestation lamb fetuses (approximately day 135 of gestation) when it is approximately 2-fold greater in males than in females. Exposure of genetic female fetuses to exogenous testosterone during the critical period for sexual differentiation masculinizes oSDN volume and aromatase expression when examined subsequently on day 135. The demonstration that the oSDN is organized prenatally by testosterone exposure suggests that the brain of the male-oriented ram may be under-androgenized during development.

Prenatal programming of sexual partner preference: the ram model.

In our laboratory, the domestic ram is used as an experimental model to study the early programming of neural mechanisms underlying same-sex partner preference. This interest developed from the observation that approximately 8% of domestic rams are sexually attracted to other rams (male-oriented) in contrast to the majority of rams that are attracted to oestrous ewes (female-oriented). One prominent feature of sexual differentiation in many species is the presence of a sexually dimorphic nucleus (SDN) in the preoptic/anterior hypothalamus that is larger in males than in females. Lesion studies in rats and ferrets implicate the SDN in the expression of sexual preferences. We discovered an ovine SDN (oSDN) in the preoptic/anterior hypothalamus that is smaller in male- than in female-oriented rams and similar in size to the oSDN of ewes. Neurones of the oSDN show abundant aromatase expression that is also reduced in male-oriented compared to female-oriented rams. This observation suggests that sexual partner preferences are neurologically hard-wired and could be influenced by hormones. Aromatase-containing neurones constitute a nascent oSDN as early as day 60 of gestation, which becomes sexually dimorphic by day 135 of gestation when it is two-fold larger in males than in females. Exposure of fetal female lambs to exogenous testosterone from days 30-90 of gestation resulted in a masculinised oSDN. These data demonstrate that the oSDN develops prenatally and may influence adult sexual preferences. Surprisingly, inhibition of aromatase activity in the brain of ram foetuses during the critical period did not interfere with defeminisation of adult sexual partner preference or oSDN volume. These results fail to support an essential role for neural aromatase in the sexual differentiation of sheep brain and behaviour. Thus, we propose that oSDN morphology and male-typical partner preferences may instead be programmed through an androgen receptor mechanism not involving aromatisation.

The volume of the ovine sexually dimorphic nucleus of the preoptic area is independent of adult testosterone concentrations.

The ovine sexually dimorphic nucleus (oSDN) is characterized by high levels of aromatase mRNA expression which can be used to delineate its boundaries. The volume of the oSDN is approximately 2 to 3-fold larger in rams that mate with ewes (female-oriented rams) than in rams that mate with other rams (male-oriented rams) and ewes. The sex difference in oSDN volume is present in late gestation fetuses and can be eliminated before birth by exposing genetic females to exogenous testosterone during midgestation, suggesting that early exposure to androgen masculinizes volume of the oSDN. The present study was performed to determine whether differences in oSDN volume are influenced by the adult hormonal environment. Adult rams, behaviorally characterized as female-oriented or male-oriented, and ewes were gonadectomized and treated with subcutaneous implants of testosterone to achieve physiologic concentrations of serum testosterone. Three weeks after implant placement brain tissue was prepared for histological assessment of oSDN volume using in situ hybridization for detection of aromatase mRNA expression. Quantitative analysis revealed that despite similar serum testosterone levels among the groups, the volume of the oSDN was greater in female-oriented rams than in male-oriented rams and ewes (P<0.05). Differences in oSDN volume were specific and not reflective of differences in preoptic area height or brain size. These results suggest that differences in the size of the oSDN in adult sheep were not influenced by adult exposure to testosterone.

Changes in LH secretion in response to an estradiol challenge in male- and female-oriented rams and in ewes.

Two experiments were conducted to determine whether an estradiol challenge could cause a female-type LH surge in castrated male- and female-oriented rams (MORs and FORs). Administration of 17beta-estradiol to castrated MORs and FORs and ovariectomized ewes caused an initial reduction in LH secretion followed for 12-20 h by a surge release of LH in the ewes. No surge release of LH occurred in the MORs and FORs. The pattern of changes in LH secretion within rams and ewes did not differ between the breeding and nonbreeding seasons. Treatment failed to elicit female-typical receptive sexual behaviors in the rams but did stimulate increased sexual receptivity in the ewes as determined by the measures of responsiveness to the teaser ram. Overall, no differences were found in hypothalamic-hypophyseal function in response to exogenous estradiol between MORs and FORs. These data are interpreted to suggest that in contrast to sexual attraction, the neural mechanisms controlling the LH surge and female receptivity are defeminized in MORs.

Non-genomic actions of progesterone and estrogens in regulating reproductive events in domestic animals.

It has been established that nuclear receptors mediate the action of estrogens and progestins in regulating gene expression in the hypothalamic-hypophyseal-gonadal axis of domestic animals during various reproductive states. Results of recent in vitro studies suggest that estradiol-17beta and progesterone can act non-genomically to affect signal transduction responses in target cells by binding to receptors in the plasma membrane. The genomic action of steroids is generally detectable in hours to days whereas non-genomic responses of cells occur in seconds to minutes. The nature of the plasma membrane receptors for estrogens and progesterone has been explored but has not been conclusively established for all cell types studied. In the ewe, estradiol-17beta or estradiol-bovine serum albumin conjugate has been shown by in vitro and in vivo approaches to act non-genomically to suppress luteinizing hormone secretion by gonadotropes and stimulate production of nitric oxide by uterine arterial endothelial cells. Progesterone has been shown to inhibit oxytocin (OT) binding to its receptor in isolated ovine endometrial plasma membranes. This non-genomic action of progesterone blocks OT activation of the phosphoinositide cascade and production of prostaglandin F(2alpha) by ovine and bovine endometrium. The acrosome reaction of caprine and porcine spermatozoa is activated by the non-genomic action of progesterone. Further research is required to define the biological significances of the non-genomic actions of estrogens and progestins.

Board-invited review: Estrogen and progesterone signaling: genomic and nongenomic actions in domestic ruminants.

Progesterone and estrogens play key roles in regulating various physiological phenomena related to normal growth, development, and reproduction of domestic animals. This review focuses on the mechanisms by which progesterone and estrogens regulate the reproductive processes in these animals. The majority of research on the actions of progesterone and estrogens on the reproductive systems of cattle, sheep, and pigs has been genomic in nature and represents attempts to better understand how these steroids regulate gene expression. Results of recent research suggest that progesterone and estrogens can alter target cell responses nongenomically via membrane receptors. The characteristics of membrane receptors for progesterone and estrogen in various cell types are described and the intracellular signal pathways defined. Estrogens acting via membrane receptors can suppress LH secretion by gonadotropes and stimulate rapid increases in uterine blood flow. Progesterone acting via a membrane receptor has been shown to inhibit binding of oxytocin to oxytocin receptors in isolated endometrial plasma membranes and stimulate capacitation of spermatozoa. Results of research suggest that progesterone and estrogens can act nongenomically to alter target cell responses in domestic animals. The biological implications of this mode of action in these animals are discussed.

The effect of aromatase inhibition on the sexual differentiation of the sheep brain.

This study tested the hypothesis that aromatization of testosterone to estradiol is necessary for sexual differentiation of the sheep brain. Pregnant ewes (n = 10) were treated with the aromatase inhibitor 1,4,6- androstatriene-3,17-dione (ATD) during the period of gestation when the sheep brain is maximally sensitive to the behavior-modifying effects of exogenous testosterone (embryonic d 50-80; 147 d is term). Control (n = 10) ewes received vehicle injections. Fifteen control lambs (7 males and 8 females) and 17 ATD-exposed lambs (7 males and 10 females) were evaluated for sexually dimorphic behavioral and neuroendocrine traits as adults. Prenatal ATD exposure had no significant effect on serum concentrations of androgen at birth, growth rates, expression of juvenile play behaviors, or the onset of puberty in male and female lambs. Rams exposed to ATD prenatally exhibited a modest, but significant, decrease in mounting behavior at 18 mo of age. However, prenatal ATD exposure did not interfere with defeminization of adult sexual partner preferences, receptive behavior, or the LH surge mechanism. In summary, our results indicate that aromatization is necessary for complete behavioral masculinization in sheep. However, before we can conclude that aromatization does not play a role in defeminization of the sheep brain, it will be necessary to evaluate whether intrauterine exposure of male fetuses to higher doses of ATD for a more extended period of time can disrupt normal neuroendocrine and behavioral development.

Spatiotemporal interactions of myristoylated alanine-rich C kinase substrate (MARCKS) protein with the actin cytoskeleton and exocytosis of oxytocin upon prostaglandin F2alpha stimulation of bovine luteal cells.

In the bovine corpus luteum (CL) phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) protein in response to prostaglandin F2alpha (PGF2alpha) is correlated with the secretion of oxytocin. The present study was conducted to 1) examine the intracellular translocation characteristics of wild-type and mutant forms of a green fluorescent protein (GFP)-conjugated MARCKS (MARCKS-GFP) after PGF2alpha treatment and 2) evaluate PGF2alpha-induced temporal changes in MARCKS-GFP and actin cortex associated with exocytosis of oxytocin. In experiment 1, cells of the bovine CL were cultured on coverslips overnight. Then, wild-type and mutant MARCKS-GFP constructs were transfected separately into cells and expression was detected through fluorescence microscopy. Forty-eight hours after transfection, cells were treated with vehicle, PGF2alpha (56 nM), or a phorbol ester (12-O-tetradecanoylphorbol-13-acetate [TPA], 1 microM). Treatment of cells expressing wild-type MARCKS-GFP with PGF2alpha and TPA resulted in translocation of MARCKS from the plasma membrane to the cytoplasm within 2.5 min. Phosphorylation mutant MARCKS-GFP (m3) protein was localized on the plasma membrane, and treatments did not cause its translocation to the cytoplasm. Myristoylation mutant MARCKS-GFP (G2A) was observed solely in the cytoplasm, and no changes were detected in the intracellular location of this mutant MARCKS after treatment. In experiment 2, luteal cells were transfected with one of the three MARCKS-GFP constructs. Cells were then fixed and probed sequentially for oxytocin and filamentous actin. Results revealed that only wild-type MARCKS-GFP transfected large luteal cells contained advanced signs of exocytosis (peripheral movement of oxytocin vesicles; shorter actin filaments) with translocation of MARCKS-GFP from membrane to cytoplasm in response to PGF2alpha treatment. These data demonstrate that phosphorylation of membrane-bound MARCKS protein is requisite for exocytosis of oxytocin to occur in bovine large luteal cells.

Ovarian function in ewes after treatment with mifepristone early during the oestrous cycle.

The aim of this study was to determine whether endogenous progesterone regulates synthesis and secretion of luteal oxytocin. In Expt 1, mature ewes (n = 5 per group) were assigned randomly to control or mifepristone (RU486) treatment groups. Ewes were injected s.c. twice a day with vehicle or 10 mg RU486 on days 5-7 of the oestrous cycle (oestrus = day 0). On day 8, after an i.v. injection with prostaglandin F(2alpha) (250 microg cloprostenol), venous blood samples were collected at frequent intervals to determine plasma oxytocin concentrations. Plasma oxytocin concentrations of RU486-treated ewes were not significantly different from those of control ewes. In Expt 2, ewes were injected s.c. each day with vehicle or 175 mg RU486 on days 2-5 of the oestrous cycle followed by administration of prostaglandin F(2alpha) on day 6. Four of five RU486-treated ewes showed 'split-oestrus' (oestrous behaviour for 36 h and then again at 84-108 h after the onset of initial oestrus). There was no significant difference in mean plasma oxytocin or progesterone concentrations between treatment groups. The mean masses of mature corpora lutea from control and RU486-treated ewes on day 6 of the oestrous cycle did not differ significantly (394.8 +/- 28.8 versus 319.5 +/- 48.3 mg). RU486-treated ewes contained mature corpora lutea, new corpora lutea (two of four ewes) and preovulatory follicles (>or= 10 mm, two of four ewes). The average interoestrous interval for RU486-treated ewes was 9 days more than that for control animals (26.2 +/- 2.9 versus 17 +/- 0.5 days; P < 0.025).

Autocrine/paracrine action of oxytocin in pig endometrium.

Luminal epithelial cells of porcine endometrium are unresponsive to oxytocin (OT) in vitro although they express the greatest quantity of OT and receptors for OT in vivo. Therefore, the objective of this study was to determine if oxytocin acted in an autocrine manner on luminal epithelial cells to stimulate prostaglandin (PG)F(2alpha) secretion. Treatment of endometrial explants or enriched luminal epithelial cells with OT antagonist L-366,948 decreased (P < 0.05) basal secretion of PGF(2alpha). Oxytocin increased (P < 0.01) PGF(2alpha) secretion from luminal epithelial cells that were pretreated with 1:5000 or 1:500 OT antiserum for 3 h to immunoneutralize endogenously secreted OT. However, OT only increased (P < 0.05) PGF(2alpha) secretion from glandular epithelial cells when pretreated with 1:500 OT antiserum. Pretreatment with OT antiserum did not alter the ability of OT to induce PGF(2alpha) secretion from stromal cells. Medium conditioned by culture of luminal epithelial cells stimulated (P < 0.05) phospholipase C activity in stromal cells, indicative of the presence of bioactive OT. Oxytocin was secreted by luminal epithelial cells and 33% was released from the apical surface. These results indicate that luminal epithelial cells secrete OT that acts in an autocrine and/or paracrine manner in pig endometrium to stimulate PGF(2alpha) secretion.

Prostaglandin F2alpha-activated protein kinase Calpha phosphorylates myristoylated alanine-rich C kinase substrate protein in bovine luteal cells.

Prostaglandin F2alpha (PGF2alpha)-induced secretion of oxytocin by the bovine corpus luteum involves the phosphorylation of a unique protein kinase C (PKC) substrate, myristoylated alanine-rich C kinase substrate (MARCKS) protein. This study was conducted to determine the specific PKC isoform engaged in phosphorylation of MARCKS protein in bovine luteal cells. In experiment 1, dispersed luteal cells recovered from the corpus luteum on d 8 of the estrous cycle were preincubated with [32P] orthophosphate and then exposed to PGF2alpha alone or in combination with PKC inhibitors. Autoradiography and densitometry of Western blots revealed that MARCKS protein was phosphorylated by a conventional PKC (cPKC) isoform. Experiment 2 was conducted to identify the specific cPKC isoform that phosphorylates MARCKS protein in luteal cells. Corpora lutea were removed from control and PGF2alpha-treated heifers on d 8 of the cycle, and PKC isoforms associated with membrane and cytosolic fractions were determined. Treatment with PGF2alpha increased membrane concentrations of PKCalpha within 5 min after treatment (p < 0.005). Collectively, these data suggest that phosphorylation of MARCKS protein coinciding with oxytocin secretion is mediated by PKCalpha.

Myristoylated alanine-rich C kinase substrate protein and mRNA in bovine corpus luteum during the estrous cycle.

The bovine corpus luteum contains a myristoylated alanine-rich C kinase substrate (MARCKS) protein known to crosslink actin filaments in the cytoskeletal cortex associated with the plasma membrane. We conducted experiments to determine whether concentrations of MARCKS mRNA and protein in the bovine corpus luteum varied during the estrous cycle. Using Northern blots probed with a MARCKS cDNA, we found that luteal concentrations of MARCKS mRNA were greatest on d 4, 8, and 12 and markedly reduced on d 16 of the cycle (p < 0.08). Similarly, Western blot analysis of luteal proteins revealed that concentrations of MARCKS protein were greatest on d 8 and least on d 16 of the cycle (p < 0.01). Exposure of slices from a d 8 corpus luteum to prostaglandin F2alpha (PGF2alpha) during a 10-min incubation in the presence of [32P]-ortho-phosphate resulted in enhanced phosphorylation of MARCKS in membrane and cytosolic fractions compared to that of controls. We therefore concluded that expression of the luteal MARCKS protein gene may be regulated and that PGF2alpha-induced phosphorylation of this protein is attributable to activation of protein kinase C.

Influence of castration and estrogen replacement on sexual behavior of female-oriented, male-oriented, and asexual rams.

An experiment was conducted to determine whether exogenous estradiol-17beta (E2) could restore sexual behavior in castrated rams. The protocol consisted of three sequential 6-wk periods during which rams were studied while 1) intact, 2) bilaterally castrated, or 3) implanted s.c. with two 7.6-cm silastic implants each containing 309+/-16 mg of E2. Rams (classified as female-oriented [FOR, n = 7], male-oriented [MOR, n = 7], or asexual [n = 7]) were subjected to 30-min sexual behavior tests every 2 wk during the ensuing 18 wk. Rams were observed for mounts and ejaculations using two ovariectomized, estrous ewes and two intact males secured in stanchions. Behavioral data were analyzed using the signed rank test, but asexual rams showed no sexual behavior and therefore were not evaluated statistically. Jugular blood was collected prior to castration and at the end of the 18-wk period, and testicular venous (n = 21) and arterial (n = 8) bloods were collected immediately prior to castration. Radioimmunoassay was used to quantify systemic levels of estrone (E1), E2, and testosterone (T) and testicular serum concentrations of oxytocin (OT). Mounting behavior of MOR and FOR declined after castration (P < .05 and P < .10, respectively). Castration reduced the number of ejaculations by FOR (P < .05), but not by MOR (P > .10). Mounting behavior of castrated MOR and FOR was not affected by E2 treatment relative to that observed if castrated only (P > .10). Treatment of asexual rams with E2 did not stimulate sexual behavior in these rams. There were no marked differences (P > .10) among ram groups with regard to serum concentrations of E1, E2, or T prior to castration (overall mean +/- SE, 12.8+/-.7, 7.6+/-.5, and 2,670+/-780 pg/mL, respectively) or any difference (P > .10) in systemic concentration of E1 or E2 among ram groups after rams were implanted with E2 (overall mean +/- SE, 9.7+/-.7 and 9.0 +/-.7 pg/mL, respectively). Serum concentrations of E2 after implantation of the steroid did not differ from those present while rams were intact (P > .10). Testicular venous and arterial serum concentrations of OT were low and did not differ within or between rams. These results suggest that restoration of E2 concentrations to physiological levels in castrated adult rams (regardless of sexual orientation) cannot stimulate or reestablish sexual behaviors to levels observed prior to castration.

Phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) protein is associated with bovine luteal oxytocin exocytosis.

The ruminant corpus luteum, in addition to producing progesterone, synthesizes and secretes oxytocin (OT) during the estrous cycle. Secretion of oxytocin occurs by exocytosis of membrane-encapsulated granules of this hormone. Exocytosis of oxytocin involves transport of granules through a cytoskeletal matrix including an actin cortex closely associated with the plasma membrane (PM). Actin filaments crosslinked by various proteins give rise to the structural integrity of the cortex. Myristoylated alanine-rich C kinase substrate (MARCKS), a protein specifically phosphorylated by protein kinase C (PKC), crosslinks actin filaments and anchors the actin network to the inner leaflet of the PM. There is evidence that the intact actin cortex may serve as a barrier, precluding fusion of transport vesicles with the PM. In some secretory cells, phosphorylation of MARCKS has resulted in its translocation from the PM to the cytoplasm with an associated disassembly of the actin cortex. Prostaglandin F(2alpha) (PGF(2alpha)) stimulation of the bovine corpus luteum during the midluteal phase of the estrous cycle activates PKC, which is associated with an increase in OT secretion in vivo and in vitro. Data are presented demonstrating that stimulation of bovine luteal cells with PGF(2alpha) on Day 8 of the cycle promotes rapid phosphorylation of MARCKS protein and causes its translocation from the PM to the cytoplasm and concomitant, enhanced exocytosis of OT. These data are consistent with the premise that MARCKS plays a role in the exocytotic process.

Distribution of aromatase mRNA in the ram hypothalamus: an in situ hybridization study.

The regional distribution of neurones expressing aromatase mRNA in the ram hypothalamus was examined by in situ hybridization using 33P-labelled cRNA probes. The highest amounts of hybridization signal were observed in the central part of the medial preoptic nucleus and posterior medial part of the bed nucleus of the stria terminalis. Moderate amounts of hybridization signal were observed in the anteroventral periventricular preoptic nucleus, medial preoptic nucleus and a broad band extending between the medial preoptic nucleus and bed nucleus of the stria terminalis. Low levels of hybridization signal were observed in the organum vasculosum of the lamina terminalis, anterior part of the medial preoptic nucleus, and central part of the ventromedial nucleus of the hypothalamus. The presence of aromatase mRNA within neurones of the steroid-sensitive hypothalamic circuit supports a role for aromatization in the mechanism of testosterone action on reproductive function in male sheep. The distribution of aromatase mRNA in the ovine hypothalamus was similar to that described for other vertebrate species, suggesting a high degree of functional conservation across species.