J. J. Ford

Central and peripheral administration of kisspeptin activates gonadotropin but not somatotropin secretion in prepubertal gilts

It is well established that kisspeptin signaling is necessary for the onset of puberty in laboratory animals. However, the role that kisspeptin may have in regulating puberty in large domestic animals is unknown. We tested the hypothesis that either central or peripheral infusion of kisspeptin would stimulate gonadotropin and GH secretion in prepubertal gilts. In experiment 1, prepubertal gilts were fitted with i.c.v. cannula and indwelling jugular catheters. Animals were randomly assigned to receive 0, 10, or 100 microg kisspeptin in saline. In experiment 2, prepubertal gilts, fitted with indwelling jugular catheters, randomly received 0, 1, 2.5, or 5 mg kisspeptin in saline intravenously. Serial blood samples were collected every 15 min for 3 h before and 5 h after infusions, and serum concentrations of LH, FSH, and GH were determined. Mean concentrations of LH and FSH remained at basal levels for control animals but were increased (P<0.001) for animals receiving i.c.v. infusion of kisspeptin. Area under the LH and FSH curves following i.c.v. infusion of kisspeptin increased (P<0.001) in a dose-dependent manner. Concentrations of GH were unaffected by i.c.v. treatment. Peripheral administration of kisspeptin increased (P<0.05) serum concentrations of LH but not FSH or GH. Thus, kisspeptin can activate gonadotropic but not somatotropic hormone secretion in prepubertal gilts. The present data support the concept that kisspeptin plays a role in the mechanism involved in initiating puberty in swine.

Temporal and Spatial Localization Patterns of Gata4 During Porcine Gonadogenesis

Abstract The zinc finger transcription factor Gata4, is associated with gonadal development in many species. The present study characterizes temporal and spatial localization of Gata4 throughout gonadogenesis in porcine embryos. Immunohistochemical studies illustrated that Gata4 protein is present in the coelomic epithelium prior to histological differentiation of the nascent bipotential gonad, marking the future site of both XX and XY porcine gonads. Many somatic cells of both XX and XY bipotential gonads continue to retain Gata4 immunoreactivity throughout sexual differentiation and subsequent gonadal development. Testicular cords were evident by 26 days postcoitum. Gata4 was present in Sertoli cells, identified by virtue of coexpression with Müllerian inhibiting substance and also interstitial cells including Leydig cells throughout fetal and postnatal life. Many somatic cells of the differentiating ovary including follicular cells also contained Gata4 protein throughout fetal and postnatal life. Gata4 was not present in germ cells, endothelial cells, or other undifferentiated mesenchymal cells of both XX and XY gonads. A population of Gata4-positive cells in the dorsal mesentery was continuous with the coelomic epithelium of the gonad. This localization pattern led to the hypothesis that a subpopulation of somatic cells in the dorsal mesentery moves toward the gonad. An in vitro cell migration assay demonstrated that Gata4-positive cells preferentially migrate toward explanted gonadal tissue, and morphological features of the developing gonad supported this hypothesis. This study illustrates that Gata4 is a very early marker for gonad formation, highlights species differences in temporal and spatial localization patterns, and suggests a potential role for Gata4 in the development of both XX and XY porcine gonads. Further, we suggest that mesenchymal cells of the dorsal mesentery may provide a source of somatic cells that migrate and incorporate into the gonad and contribute to various somatic cell lineages. Overall, the spatial and temporal localization patterns of Gata4 during porcine gonadogenesis implies a much earlier and wider role for Gata4 than previously reported in other species.

Growth Hormone Secretion after Hypophysial Stalk Transection in Pigs

Abstract The level and pattern of growth hormone (GH) secretion were investigated in mature ovariectomized gilts after hypophysial stalk transection and sham operation. A nylon disk was inserted between severed ends of the stalk to prevent vascular regeneration. Blood samples were collected via indwelling jugular catheters at 15-min intervals for 3 hr, 2 days before surgery (Day −2) and 2 days after surgery (Day +2), and at 4-hr intervals from Day +3 to Day +8. Mean overall serum concentrations of GH after hypophysial stalk transection remained similar (P > 0.05) to presurgical levels. These mean concentrations also were similar to those in shamoperated and unoperated controls. However, hypophysial stalk transection significantly dampened (P < 0.05) the episodic secretion of GH and resulted in elevated basal blood concentrations of the hormone as compared with either presurgical levels or those in the two control groups. These results indicate that synthesis and secretion of GH continue in the absence of hypothalamic control in hypophysial stalk-transected gilts. Thus, the hypothalamus is required for regulation of both episodic release and the tonic inhibition of basal secretion of growth hormone in the pig.

Effect of gonadotropin treatment on size, number, and cell proliferation of antral follicles in cows

To determine the effects of gonadotropins on the size, number, and cell proliferation of antral ovarian follicles, cows received FSH-P or vehicle beginning on Day 2 after estrus, and ovaries were collected 6, 12, 24, or 48 hr after the initiation of FSH-P treatment or 24 or 48 hr after the initiation of vehicle treatment. Ovaries also were collected from untreated cows on Day 2 after estrus (pretreatment). Before fixation, all visible antral follicles were counted and their surface diameters were recorded. Proliferating cells were immunolocalized in fixed follicles by using a specific primary antibody against proliferating cell nuclear antigen (PCNA), and the labeling index (LI; percentage of cells staining positively for PCNA) was determined for granulosa and thecal cells. After 48 hr of treatment, FSH-P-treated cows had fewer (P < 0.01) small antral follicles and more medium and large antral follicles (P < 0.01 and P < 0.05, respectively) compared with vehicle-treated cows. Granulosa cell LI was negatively correlated (P < 0.05) with follicular diameter for vehicle-treated but not for FSH-P-treated cows. Analysis of covariance using follicular diameter as a covariate to adjust to a common diameter indicated that granulosa cell LI was greater (P < 0.05) at 24 and 48 hr in FSH-P-treated than in vehicle-treated cows; conversely, thecal cell LI was greater (P < 0.01) at 48 hr in FSH-P-treated compared with vehicle-treated cows but did not differ at 24 hr. Across all groups, the LI of cells located within the antral half of the granulosa cell layer was greater (P < 0.01) than that of cells located within the basal half. In conclusion, the stimulation of follicular development by exogenous gonadotropins increased or maintained the proliferation of granulosa and thecal cells concomitant with continued follicular growth. Therefore, enhanced follicular cell proliferation may be an important mechanism by which FSH-P superinduces the growth of antral follicles in cows.

Biochemical Assessment of Limits to Estrogen Synthesis in Porcine Follicles

Abstract Limits to estrogen production by early and late preovulatory porcine follicles were assessed by comparing enzymatic capacities for androgen (17,20-lyase) and estrogen (aromatase) synthesis in theca interna and granulosa, support of enzyme activities by the redox partner proteins NADPH-cytochrome P450 oxidoreductase (reductase) and cytochrome b5, and tissue-specific expression and regulation of these proteins. Parameters included follicular fluid (FF) estradiol and progesterone levels, theca and granulosa aromatase and reductase activities, and theca 17,20-lyase activity. Expression of proteins responsible for these activities, aromatase (P450arom) and 17α-hydroxylase/17,20-lyase (P450c17) cytochromes P450, reductase, and for the first time in ovarian tissues cytochrome b5, were examined by Western immunoblot and immunocytochemistry. Theca and granulosa aromatase activities were as much as 100-fold lower than theca 17,20-lyase activity, but aromatase was correlated with only the log of FF estradiol. Granulosa reductase activity was twice that of the theca, and cytochrome b5 expression was clearly identified in both the theca and granulosa layers, as was P450arom, but was not highly correlated with either 17,20-lyase or aromatase activities. Reductase expression did not change with stage of follicular development, but cytochrome b5, P450c17, and P450arom were markedly lower in post-LH tissues. These data indicate that aromatase and not 17,20-lyase must limit porcine follicular estradiol synthesis, but this limitation is not reflected acutely in FF steroid concentrations. Neither reductase nor cytochrome b5 appear to regulate P450 activities, but the expression of cytochrome b5 in granulosa and theca suggests possible alternative roles for this protein in follicular development or function.

Sexual Differentiation and the Growth Process

As adults, males are larger than females in most species with which we are familiar, but this generalization is not appropriate for all species (Ralls, 1976). In cattle, sheep, and swine, testicular secretions (testosterone and its metabolites) are associated with the greater size of males, but few discussions of growth in domestic farm animals address the total impact of these steroids on developmental processes. The influence of testicular steroids on growth and muscling during pubertal development is well documented (Tucker and Merkel, 1987), but when steers are produced by castration shortly after birth, they are not exposed to testicular secretions during postnatal development. Why then do steers grow faster and larger than heifers? A second point that has perplexed animal scientists is the inconsistency among cattle, sheep, and swine relative to body growth after castration of young males. From Hammond’s Farm Animals (Hammond et al., 1971) we quote, ‘‘While (at equal body weight) the castrated male, in sheep or cattle, has a higher proportion of muscle and less fat than the female, in pigs the position is reversed.” Trenkle and Marple (1983) reiterated this point: “The inconsistent ranking of the barrow as compared with the steer and wether is not easily explained.” We also are unable to fully explain this issue but speculate that this may relate to the time when sexual differentiation of the growth process occurs.

Growth Hormone and Prolactin Secretion in Hypophysial Stalk-Transected Pigs as Affected by Growth Hormone and Prolactin-Releasing and Inhibiting Factors

Abstract Control of growth hormone (GH) and prolactin (PRL) release was investigated in hypophysial stalk-transected (HST) and stalk-intact pigs by determining the effects of analogs of GH-releasing factors (GHRF), somatostatin (SRIF), arginine, thyrotropin-releasing hormone, α-methyl-ρ-tyrosine, and haloperidol. HST and control gilts were challenged with intravenous injections of human pancreatic GHRF(1–40)OH, thyrotropin-releasing hormone, and analogs of rat hypothalamic GHRF. HST animals remained acutely responsive to GHRF by releasing 2-fold greater quantities of GH than seen in controls. This occurred in spite of a 38% reduction in pituitary gland weight and a 32 and 55% decrease in GH concentration and total content. During SRIF infusion, GH remained at similar basal concentrations in HST and control gilts, but increased immediately after stopping SRIF infusion only in the controls. Releasable pituitary GH appears to accumulate during SRIF infusion. GHRF given during SRIF infusion caused a 2-fold greater release of GH than seen in animals receiving only GHRF. Arginine increased (P < 0.05) GH release in controls, but not in HST gilts, which suggests that it acts through the central nervous system. Basal PRL concentrations were greater (P < 0.05) in HST gilts than in control gilts. TRH acutely elevated circulating PRL (P < 0.001) in HST gilts, suggesting that it acts directly on the pituitary gland. Haloperidol, a dopamine receptor antagonist, increased circulating PRL in controls but not in HST animals. α-Methyl-ρ-tyrosine did not consistently increase circulating PRL, however, suggesting that it did not sufficiently alter turnover rate of the tyrosine hydroxylase pool. The results indicate that the isolated pituitary after HST remains acutely responsive to hypothalamic releasing and inhibiting factors for both GH and PRL release in the pig.

Prenatal Androgen Exposure and Growth and Secretion of Growth Hormone and Prolactin in Ewes Postweaning

Abstract Growth and secretion of growth hormone (GH) and prolactin (PRL) in ewe lambs exposed to androgen during fetal development were investigated. Testosterone cypionate was administered to the pregnant dams from approximately Days 28 to 84 of gestation. Ewe lambs from dams that received androgen exhibited masculinized external genitalia and some masculine behavioral characteristics. Intact androgenized ewe lambs grew faster (P < 0.05) and were more efficient in conversion of food to body gain (P < 0.05) than ewe lambs born to untreated dams over the period from 70 to 224 days of age. One-half of the ewe lambs in each group was ovariectomized at 58 days of age. Ovariectomy had no effect on subsequent growth or efficiency of growth in the control ewe lambs. However, ovariectomy of androgenized ewe lambs abolished the observed stimulated rate of growth and decreased the improvement in efficiency of food conversion. Blood samples were collected from the lambs at 85 and 136 days of age at 15-min intervals for 8 hr to determine parameters of GH and PRL secretion. Prenatal androgen exposure had no effect on any parameter of GH or PRL secretion. These data indicate that prenatal androgen exposure altered differentiation of growth potential in ewe lambs, but the growth response was not mediated through dramatic changes in secretion of adenohypophysial somatotropic hormones, GH and PRL.

Germ cell development in Meishan and White Composite gilts

This study compared dynamics of the germ cell population in two swine breeds that differ in prolifacy, White Composite (WC) and Meishan (MS), during fetal and neonatal life and in mature sows. Germ cell populations developed in a similar pattern in these two diverse breeds during fetal life. Maximal germ cell number was observed at 90 days postcoitum (dpc) in both WC and MS gilts, and substantial oogonial apoptosis was evident thereafter with approximately 30% of maximal numbers present at 25 days postpartum (dpp). Neither gilt nor sow germ cell number was correlated with maternal ovulation rate. Postnatal MS gilts had larger pools of primordial follicles and consistently greater proportions and numbers of primary and secondary follicles compared to postnatal WC gilts, indicative of enhanced follicular recruitment and primordial follicle activation. Occasional antral follicles were present in MS ovaries by 25 dpp and numerous surface follicles were observed at 56 dpp in MS but not WC ovaries, indicative of more rapid ovarian maturation and early onset of puberty. Total germ cell number is unlikely to influence or to predict subsequent ovulation rate. These observations highlight important developmental events during late fetal and early postnatal life that prepare the ovarian environment for early onset of puberty and subsequent ovulation in MS gilts.

Growth hormone and prolactin secretion after hypothalamic deafferentation in pigs.

Abstract Control of growth hormone (GH) and prolactin (PRL) secretion was investigated in ovariectomized, prepuberal Yorkshire gilts by comparing the effects of anterior (AHD), complete (CHD), and posterior (PHD) hypothalamic deafferentation with sham-operated controls (SOC). Blood samples were collected sequentially via an indwelling jugular catheter at 20-min intervals during surgery and recovery from anesthesia (Day 0) and Days 1 and 2 after cranial surgery. Mean serum concentrations of GH after AHD, CHD, and PHD were reduced (P < 0.01) when compared with SOC gilts. Furthermore, episodic GH release evident in SOC animals was obliterated after hypothalamic deafferentation. PRL concentrations in peripheral serum of hypothalamic deafferentated gilts remained similar (P > 0.05) to those of SOC animals. These results indicate that anterior and posterior hypothalamic neural pathways play a minor role in the control of PRL secretion in the pig in as much as PRL levels remained unchanged after hypothalamic deafferentation. These findings may be interpreted to suggest that the hypothalamus by itself seems able to maintain tonic inhibition of PRL release. In contrast, the maintenance of episodic GH secretion depends upon its neural connections traversing the anterior and posterior aspects of the hypothalamus in the pig.