Heywood R. Sawyer

Formation of Ovarian Follicles During Fetal Development in Sheep

Abstract The origin of follicle (i.e., pregranulosa) cells that become the somatic component of primordial follicles is obscure. In addition, information regarding the structural changes that accompany the concomitant regression of ovigerous cords and the appearance of primordial follicles is lacking. In the present study, ovine ovaries collected at frequent time intervals between Day 38 and Day 100 of fetal life were examined by light and electron microscopy. To gain new information regarding the origin of follicular cells, incorporation of 5-bromo-2′-deoxyuridine was used to identify proliferating cells at selected stages of development. Based on the location and identity of proliferating cells, apoptotic cells, and sequential changes in histoarchitecture, we hypothesize 1) that most (i.e., >95%) of the granulosal cells in newly formed primordial follicles originate from the ovarian surface epithelium; 2) that the sequential events leading to follicle formation take place entirely within ovigerous cords, with the first follicles forming at the interface of the cortex and medulla; and 3) that the loss (i.e., >75%) of germ cells, but not of somatic cells, within the ovigerous cords is a means by which each surviving oocyte gains additional pregranulosal cells before follicle formation. Conceptual models detailing the chronology of developmental events involved in the formation of primordial follicles in sheep are discussed.

Expression of Growth and Differentiation Factor-9 in the Ovaries of Fetal Sheep Homozygous or Heterozygous for the Inverdale Prolificacy Gene (FecXI)

Abstract Abnormal follicular and oocyte growth in ovaries of sheep homozygous (II) for the Inverdale gene, FecXI, suggest that this gene may influence a fundamental event in initiation of folliculogenesis, with two copies of the gene inhibiting growth at the primordial/primary stage. In addition, striking similarities in ovarian morphology between mice deficient in growth and differentiation factor-9 (GDF-9) and II sheep suggest a relationship between the FecXI gene and GDF-9 function in the ovary. Therefore, it was hypothesized that GDF-9 mRNA expression would be inhibited in ovaries of II fetal sheep. To test this hypothesis, in situ hybridization was used to characterize GDF-9 mRNA expression in ovaries of homozygous (II), heterozygous (I+), and control (++) fetal sheep at Day 135 of gestation. GDF-9 mRNA expression was localized exclusively to oocytes from the type 1 follicle stage onward in all genotypes and is the first demonstration of GDF-9 mRNA expression in ovaries of fetal sheep. In addition, GDF-9 mRNA expression was detected in oocytes of abnormal type 2 follicles in the ovaries of II sheep. Thus, it does not appear that inhibition of GDF-9 gene expression is the mechanism of action whereby the FecXI gene exerts its influence. However, the possibility of translation at specific stages of follicular development cannot presently be ruled out. In addition, the FecXI gene may be involved, either directly or indirectly, in regulating expression of receptors for GDF-9. At present, however, neither the FecXI gene product nor the GDF-9 receptor has been isolated or characterized.

Effect of Dose of Prostaglandin F2α on Steroidogenic Components and Oligonucleosomes in Ovine Luteal Tissue

Abstract To determine whether prostaglandin (PG) F2α had a dose-dependent effect upon secretion of progesterone, oligonucleosome formation, or loss of luteal weight, ewes on Day 9 or 10 of the estrous cycle were administered 0, 3, 10, or 30 mg PGF2α per 60 kg BW (i.v.), and luteal tissue was collected 9 and 24 h after injection. All doses of PGF2α decreased (P < 0.05) concentrations of progesterone in sera by 9 h; however, in ewes treated with 3 mg PGF2α, concentrations of progesterone were similar to control values at 24 h and higher (P < 0.05) than those in the 10- or 30-mg groups. Concentrations of progesterone in sera over all dose levels were highly correlated to luteal concentrations of mRNA encoding steroidogenic acute regulatory protein (P < 0.001), cytochrome P450 side-chain cleavage (P < 0.02), and 3β-hydroxysteroid dehydrogenase (P < 0.01). Corpora lutea collected at 24 h from ewes treated with the 10- and 30-mg doses of PGF2α weighed less (P < 0.05) than those from controls. Oligonucleosomes were not present in luteal tissues from control ewes. Surprisingly, all doses of PGF2α-induced oligonucleosomes in a majority of animals at 9 h and in a majority of ewes treated with 10 and 30 mg of PGF2α at 24 h. In conclusion, 3 mg of PGF2α per 60 kg BW transiently decreased serum concentrations of progesterone and induced oligonucleosome formation, but did not result in reduced luteal weight. The 10- and 30-mg doses of PGF2α decreased secretion of progesterone and induced oligonucleosome formation and luteolysis.

PGE2 attenuates PGF2α-induced increases in free intracellular calcium in ovine large luteal cells

When ovine large luteal cells are placed in culture and exposed to PGF2 alpha, there is a rapid and sustained increase in the concentration of free intracellular calcium which is believed to play a major role in the luteolytic and cytotoxic effects of PGF2 alpha. Since administration of exogenous PGE2 can prevent spontaneous and PGF2 alpha-induced luteolysis in vivo, and the cytotoxic effects of PGF2 alpha on large luteal cells in vitro, the objective of this study was to determine if one mechanism by which PGE2 acts is to attenuate increases in free intracellular calcium induced by PGF2 alpha. At concentrations of 10 nM or greater, PGF2 alpha caused a significant and sustained increase in free intracellular calcium in large luteal cells. Similarly, PGE2 also induced increases in free intracellular calcium but required doses 20-fold greater than PGF2 alpha. When PGE2 (1, 10 or 100 nM) was incubated with PGF2 alpha (100 nM) increases in free intracellular calcium induced by PGF2 alpha were attenuated (P < 0.05) when measured 5 min, but not at 30 min, after initiation of treatment. The observed decrease in the concentration of free intracellular calcium at 5 min in response to PGF2 alpha was the result of fewer cells responding to PGF2 alpha. In addition, the concentrations of free intracellular calcium attained in the cells that did respond was reduced 25% compared to cells treated with PGF2 alpha alone. Thus, part of the luteal protective actions of PGE2 appears to involve an inhibition of the early (5 min) increase in free intracellular calcium induced by PGF2 alpha.

Carcinoma in situ and seminoma in equine testis

The presence of atypical germ cells resembling carcinoma in situ of human testis is reported for the first time in an unilaterally cryptorchid stallion. These cells were found in association with developing intratubular seminoma indicating they represented carcinoma in situ.

Use of Semen as Biopsy Material for Assessment of Health Status of the Stallion Reproductive Tract

Conventional light microscopic evaluation does not fully utilize potential indicators in seminal ejaculates for diagnosis of disorders of the reproductive tract. The technique of evaluation of all cellular components of semen, as described in this article, utilizing both light and transmission electron microscopy is a valuable diagnostic tool. Compare with other common biopsy procedures, use of semen as biopsy material is noninvasive, more representative than excisional biopsy, less expensive, and helps in the longitudinal evaluation after a therapeutic regimen.

Effects of luteinizing hormone and growth hormone on luteal development in hypophysectomized ewes

To test the hypothesis that growth hormone (GH) as well as luteinizing hormone (LH) is required for normal luteal growth and function, 16 western range ewes were hypophysectomized (HPX) on day 5 of the estrous cycle. Ewes were randomly assigned to receive saline (S), LH, GH, or LH + GH (n=4 per group) from the time of HPX until collection of corpora lutea 7 days after HPX (day 12). Corpora lutea were also collected from pituitary-intact ewes on days 5 (day 5 control,n=4) and 12 (day 12 control,n=4) of the estrous cycle. To assess luteal function, concentrations of progesterone in sera, luteal weights and luteal concentrations of mRNA encoding cytochrome P450 side-chain cleavage enzyme (P450scc) and 3β-hydroxysteroid dehydrogenase/Δ5,Δ4 isomerase (3β-HSD) were determined. Concentrations of progesterone in sera and luteal weights increased between days 5 and 12 of the estrous cycle in control ewes, but not in HPX + S ewes. In HPX ewes treated with LH, concentrations of progesterone in sera and luteal mRNA for P450scc and 3β-HSD increased but luteal weights were unaffected. Treatment with GH increased luteal weight and luteal concentrations of mRNA encoding P450scc but did not increase concentrations of mRNA encoding 3β-HSD compared to HPX + S ewes. Concentrations of progesterone in sera of GH-treated, HPX ewes were similar to those of day 12 control ewes but not significantly different from those in HPX + S ewes. Treatment of HPX ewes with LH + GH increased all parameters of luteal function measured to values similar to those in day 12 controls. In conclusion, both GH and LH are necessary for normal luteal development in the ewe.