Descriptions of organosulfurs altering biologically relevant cellular functions began some 40 years ago when murine in vitro cell mediated and humoral immune responses were shown to be dramatically enhanced by any of four xenobiotic, sulfhydryl compounds—2-mercaptoethanol (2ME), dithiothreitol (DTT), glutathione, and L-cysteine; the most effective were 2ME and DTT. These findings triggered a plethora of reports defining 2ME benefits for a multitude of immunological processes. This in turn led to investigations on 2ME alterations of (a) immune functions in other species, (b) activities of other cell-types, and (c) in vivo diseases. In addition, these early findings preceded the identification of previously undefined anticarcinogenic chemicals in specific foods as organosulfurs. Taken all together, there is little doubt that organosulfur compounds have enormous benefits for cellular functions and for a multitude of diseases. Issues of importance still to be resolved are (a) clarification of mechanisms that underlie alteration of in vitro and in vivo processes and perhaps more importantly, (b) which if any in vitro alterations are relevant for (i) alteration of in vivo diseases and (ii) identification of other diseases that might therapeutically benefit from organosulfurs. As one means to address these questions, reviews of different processes impacted by thiols could be informative. Therefore, the present review on alterations of in vitro fertilization processes by thiols (mainly 2ME, since cysteamine alterations have been reviewed) was undertaken. Alterations found to occur in medium supplemented with 2ME were enhancement, no effect, or inhibition. Parameters associated with which are discussed as they relate to postulated thiol mechanisms.
A series of experiments was conducted to investigate migration, proliferation and differentiation of gonadal germ cells (GGCs) collected from the gonads of 7-day-old chick embryos under cross-sex germline chimera conditions. The migratory and proliferative abilities of exogenous GGCs were examined by transferring 50 fluorescently labeled GGCs collected from White Leghorn (WL) embryos into the blood of 2-day-old Rhode Island Red (RIR) embryos. No significant difference was observed in the number of fluorescently labeled GGCs in the gonads of recipient embryos among any of the four possible donor and recipient sex combinations. Cross-sex germline chimeras were produced to examine the differentiation of GGCs by transferring 100 GGCs from WL embryos into 2-day-old RIR embryos. Exogenous-GGC-derived progeny were obtained from both male and female recipients, except when female GGCs were transferred into male recipients. The migratory ability of GGCs recovered from the 7-day-old embryonic gonad was not influenced by cross-sex germ cell transfer conditions, whereas the differentiation of the GGCs was affected by the sex combinations of GGCs donors and recipients.
In vitro maturation of vitrified immature germinal vesicle (GV) oocytes is a promising fertility preservation option. We analyzed the ultrastructure of human GV oocytes after Cryotop vitrification (GVv) and compared it with fresh GV (GVc), fresh mature metaphase II (MIIc) and Cryotop-vitrified mature (MIIv) oocytes. By phase contrast microscopy and light microscopy, the oolemmal and cytoplasmic organization of fresh and vitrified oocytes did not show significant changes. GVv oocytes showed significant ultrastructural alterations of the microvilli in 40% of the samples; small vacuoles and occasional large/isolated vacuoles were abnormally present in the ooplasm periphery of 50% of samples. The ultrastructure of nuclei and mitochondria-vesicle (MV) complexes, as well as the distribution and characteristics of cortical granules (CGs), were comparable with those of GVc oocytes. MIIv oocytes showed an abnormal ultrastructure of microvilli in 30% of the samples and isolated large vacuoles in 70% of the samples. MV complexes were normal, but mitochondria-smooth endoplasmic reticulum aggregates appeared to be of reduced size. CGs were normally located under the oolemma but presented abnormalities in distribution and matrix electron density. In conclusion, Cryotop vitrification preserved main oocyte characteristics in the GV and MII stages, even if peculiar ultrastructural alterations appeared in both stages. This study also showed that the GV stage appears more suitable for vitrification than the MII stage, as indicated by the good ultrastructural preservation of important structures that are present only in immature oocytes, like the nucleus and migrating CGs.
Feral raccoons (Procyon lotor) have been growing in number in Japan, and they are becoming a problematic invasive species. Consequently, they are commonly captured and killed in pest control programs. For effective population control of feral raccoons, it is necessary to understand their reproductive physiology and ecology. Although the reproductive traits of female raccoons are well known, those of the males are not well understood because specialized knowledge and facilities are required to study them. In this study, we first used a simple evaluation method to assess spermatogenesis and presence of spermatozoa in the tail of the epididymis of feral male raccoons by histologically examining the testis and epididymis. We then evaluated the possibility of using 7 variables—body weight, body length, body mass index, testicular weight, epididymal weight, testicular size and gonadosomatic index (GSI)—to estimate spermatogenesis and presence of spermatozoa in the tail of the epididymis. GSI and body weight were chosen as criteria for spermatogenesis, and GSI was chosen as the criterion for presence of spermatozoa in the tail of the epididymis. Because GSI is calculated from body weight and testicular weight, this model should be able to be used to estimate the reproductive state of male raccoons regardless of season and age when just these two parameters are known. In this study, GSI was demonstrated to be an index of reproductive state in male raccoons. To our knowledge, this is the first report of such a use for GSI in a member of the Carnivora.
This study compares in two experiments the responses of lactating dairy cows to four different progesterone-based protocols for fixed-time artificial insemination (FTAI) in terms of their effects on follicular/luteal dynamics and fertility. The protocols consisted of a progesterone intravaginal device fitted for five days, along with the administration of different combinations of gonadotropin releasing hormone, equine chorionic gonadotropin and a single or double dose (24 h apart) of prostaglandin F2α. In Experiment I, the data were derived from 232 lactating cows. Binary logistic regression identified no effects of treatment on ovulation failure or multiple ovulation 10 days post artificial insemination (AI). Based on the odds ratio, the likelihood of ovulation failure was lower (by a factor of 0.1) in cows showing at least one corpus luteum (CL) upon treatment than in cows lacking a CL; repeat breeders (> 3 AI) and cows with multiple CLs at treatment showed lower (by a factor of 0.44) and higher (by a factor of 9.0) risks of multiple ovulation, respectively, than the remaining animals. In Experiment II, the data were derived from 5173 AIs. The independent variable treatment failed to affect the conception rate 28–34 days post AI, twin pregnancy or early fetal loss 58–64 days post AI. The results of this study demonstrate the efficacy of 5-day progesterone-based protocols for FTAI. All four protocols examined were able to induce ovulation in both cyclic and non-cyclic animals so that FTAI returned a similar pregnancy rate to spontaneous estrus. Our results suggest that the ovarian response and fertility resulting from each treatment are due more to the effect of ovarian structures at treatment than to the different combinations of hormones investigated.
The aim of the present study was to examine the effect of the somatic cell count (SCC) in milk on reproductive performance, such as pregnancy status in the prepartum period and ovarian function in the postpartum period, in dairy cows. Blood samples were collected every week from one month prepartum to parturition in order to measure the concentrations of 13,14-dihydro-15-keto-PGF2α (PGFM), estrone sulfate (E1S) and progesterone. Milk samples were collected three times per week in both the prepartum (for one month before the dry period) and postpartum periods (for 3 months immediately after parturition) to measure the SCC. Progesterone was also determined in the whole milk of postpartum cows to define the day of the first ovulation. In the prepartum period, the maximum SCC negatively correlated with the pregnancy period (r = –0.77), but not the calf birth weight. Positive and negative correlations were observed between the average SCC and PGFM or progesterone concentrations in plasma, respectively (r = 0.84 or –0.92, respectively), at 39 weeks of pregnancy. In the postpartum period, a correlation was observed between the day of the first ovulation and both the average and maximum SCC (r = –0.74 and –0.75, respectively), whereas days open was not related to the SCC. These results suggest that a high SCC in the prepartum period may advance parturition by increasing PGF2α and decreasing progesterone and that the first ovulation in the postpartum period was affected by a high SCC.
We have determined uterine glycogen content, metabolizing enzyme expression and activity in the mink, a species that exhibits obligatory embryonic diapause, resulting in delayed implantation. Gross uterine glycogen concentrations were highest in estrus, decreased 50% by diapause and 90% in pregnancy (P ≤ 0.05). Endometrial glycogen deposits, which localized primarily to glandular and luminal epithelia, decreased 99% between estrus and diapause (P ≤ 0.05) and were nearly undetectable in pregnancy. Glycogen synthase and phosphorylase proteins were most abundant in the glandular epithelia. Glycogen phosphorylase activity (total) in uterine homogenates was higher during estrus and diapause, than pregnancy. While glycogen phosphorylase protein was detected during estrus and diapause, glycogen synthase was almost undetectable after estrus, which probably contributed to a higher glycogenolysis/glycogenesis ratio during diapause. Uterine glucose-6-phosphatase 3 gene expression was greater during diapause, when compared to estrus (P ≤ 0.05) and supports the hypothesis that glucose-6-phosphate resulting from phosphorylase activity was dephosphorylated in preparation for export into the uterine lumen. The relatively high amount of hexokinase-1 protein detected in the luminal epithelia during estrus and diapause may have contributed to glucose trapping after endometrial glycogen reserves were depleted. Collectively, our findings suggest to us that endometrial glycogen reserves may be an important source of energy, supporting uterine and conceptus metabolism up to the diapausing blastocyst stage. As a result, the size of uterine glycogen reserves accumulated prior to mating may in part, determine the number of embryos that survive to the blastocyst stage, and ultimately litter size.
Angiogenesis in the developing corpus luteum (CL) is a prerequisite for establishment and maintenance of an early pregnancy. To explore the physiological significance of insulin-like growth factor-binding protein-7 (IGFBP7) in the developing CL, the effects of IGFBP7 on vascular endothelial growth factor (VEGFA)- and luteinizing hormone (LH)-induced in vitro tube formation were tested using isolated luteal microvascular endothelial cells (LECs). Capillary-like tube formation of LECs and their proliferation were stimulated by both VEGFA and LH. IGFBP7 treatment suppressed VEGFA- or LH-induced tube formation. The proliferation and migration of LECs, and phosphorylation of mitogen-activated protein kinase kinase and extracellular signal-regulated kinase 1/2 were inhibited by IGFBP7. Furthermore, IGFBP7 attenuated VEGFA-enhanced cyclooxygenase (COX)-2 mRNA expression and prostaglandin E2 secretion. These findings suggest the possibility that luteal IGFBP7 secretion may suppress the stimulatory effect of VEGFA on angiogenesis in the early CL.
Hyaluronidase is generally used to remove cumulus cells from mouse oocytes before oocyte cryopreservation, intracytoplasmic sperm injection or DNA injection. In general, use of cumulus-free mouse oocytes decreases in vitro fertilizing ability compared with cumulus-surrounded oocytes. The effect of hyaluronidase exposure on the quality of mouse oocytes is not fully understood. Here, we investigated the effect of hyaluronidase exposure time on the fertilization rate of fresh and vitrified mouse oocytes and their subsequent developmental ability in vitro. We found that the fertilization rate decreased with hyaluronidase treatments. This reduction in the fertilization rate following treatment with hyaluronidase was fully reversed by removal of the zona pellucida. In addition, oocytes treated with hyaluronidase for 5 min or longer had a reduced capacity to develop to the morula and blastocyst stage. The survival, fertilization, and developmental rates of vitrified-warmed oocytes were also reduced by longer exposure to hyaluronidase. In conclusion, these results suggest that prolonged exposure to hyaluronidase decreases the quality of mouse oocytes and shorter hyaluronidase treatment times may help achieve a stable and high fertilization rate in fresh and cryopreserved oocytes.
The study of human ovarian tissue transplantation and cryopreservation has advanced significantly. Autotransplantation of human pre-antral follicles isolated from cryopreserved cortical tissue is a promising option for the preservation of fertility in young cancer patients. The purpose of the present study was to reveal the effect of vitrification after low-temperature transportation of human pre-antral follicles by using the oxygen consumption rate (OCR). Cortical tissues from 9 ovaries of female-to-male transsexuals were vitrified after transportation (6 or 18 h). The follicles were enzymatically isolated from nonvitrified tissue (group I, 18 h of transportation), vitrified-warmed tissue (group II, 6 and 18 h of transportation) and vitrified-warmed tissue that had been incubated for 24 h (group III, 6 and 18 h of transportation). OCR measurement and the LIVE/DEAD viability assay were performed. Despite the ischemic condition, the isolated pre-antral follicles in group I consumed oxygen, and the mean OCRs increased with developmental stage. Neither the transportation time nor patient age seemed to affect the OCR in this group. Meanwhile, the mean OCR was significantly lower (P < 0.05) in group II but was comparable to that of group I after 24 h of incubation. The integrity of vitrified-warmed primordial and primary follicles was clearly corroborated by the LIVE/DEAD viability assay. These results demonstrate that the OCR can be used to directly estimate the effect of vitrification on the viability of primordial and primary follicles and to select the viable primordial and primary follicles from vitrified-warmed follicles.
The forkhead box a (Foxa) protein family has been found to play important roles in mammals. Recently, the expression of Foxa2 was reported in the mouse uterus, and it was reported to be involved in regulation of implantation. However, the regulation of Foxa2 expression in the uterus is still poorly understood. Therefore, the present study was conducted to investigate the expressional profiles of Foxa2 in the rat uterus during the estrus cycle and pregnancy. Furthermore, the effect of steroid hormones and Hedgehog protein on the expression of Foxa2 was analyzed in vivo and in vitro. In this study, the level of expression of Foxa2 was low in the rat uterus during the different stages of the estrus cycle. However, the expression increased transiently during early pregnancy at 3.5 days post coitus (dpc) and decreased at 5.5 dpc. In ovariectomized rats, P4 treatment had no effect on the expression of Foxa2 compared with the expression in control animals. Moreover, the expression of Foxa2 in cultured epithelial cells was not increased by P4 treatment in vitro. However, Foxa2 expression was significantly decreased in the rat uterus after 24 h of E2 treatment. Treatment of cells with a recombinant Hedgehog protein significantly increased the expression of Foxa2. These results suggest that the expression of Foxa2 may transiently increase just before the implantation and it may be regulated by E2 and Hedgehog protein.
Nineteen cycling ewes underwent transrectal ultrasonography of ovaries followed by ovariectomies during the growth phase of the first follicular wave of the interovulatory interval or the proestrus/estrus phase of the cycle. Quantitative ultrasonographic characteristics of the antrum and follicular wall in a total of forty-three ovine antral follicles were examined for correlations with the protein expression of three steroidogenic enzymes (cytochrome P450 17α-hydroxylase, CYP17; cytochrome P450 aromatase, CYP19; and 3β-hydroxysteroid dehydrogenase, 3β-HSD) determined by densitometric analysis of immunohistochemical slides, follicular dimensions, granulosa layer thickness and the percentage of apoptotic granulosa cells. Significant correlations were found between echotextural attributes of ovine antral follicles and the percentage of apoptotic granulosa cells, CYP17 expression (theca), CYP19 expression (granulosa) and 3β-HSD expression (theca cells). Computer-aided analyses of ultrasonographic images can be beneficial to the development of assisted reproductive technologies and diagnosis of hormonal imbalances without the need for ovarian biopsies or hormone assays.