SIT1 is a betaine/proline transporter that is activated in mouse eggs after fertilization and functions until the 2-cell stage.

Betaine (N,N,N-trimethylglycine) added to culture media is known to substantially improve the development of preimplantation mouse embryos in vitro, and to be imported into 1-cell embryos by a transporter that also accepts proline. Here, we found that the betaine/proline transporter is active in preimplantation mouse embryos only for a short period of development, between the 1- and 2-cell stages. Betaine/proline transport was activated after fertilization, beginning approximately 4 hours post-egg activation and reaching a maximum by approximately 10 hours. One- and 2-cell embryos contained endogenous betaine, indicating that a likely function for the transporter in vivo is the accumulation or retention of intracellular betaine. The appearance of transport activity after egg activation was independent of protein synthesis, but was reversibly blocked by disruption of the Golgi with brefeldin A. We assessed two candidates for the betaine/proline transporter: SIT1 (IMINO; encoded by Slc6a20a) and PROT (Slc6a7). mRNA from both genes was present in eggs and 1-cell embryos. However, when exogenously expressed in Xenopus oocytes, mouse PROT did not transport betaine and had an inhibition profile different from that of the embryonic transporter. By contrast, exogenously expressed mouse SIT1 transported both betaine and proline and closely resembled the embryonic transporter. A morpholino oligonucleotide designed to block translation of SIT1, when present from the germinal vesicle stage, blocked the appearance of betaine transport activity in parthenogenotes. Thus, SIT1 is likely to be a developmentally restricted betaine transporter in mouse preimplantation embryos that is activated by fertilization.

The organic osmolytes betaine and proline are transported by a shared system in early preimplantation mouse embryos.

Betaine and proline protect preimplantation mouse embryos against increased osmolarity and decreased cell volume, implying that they may function as organic osmolytes. However, the transport system(s) that mediates their accumulation in fertilized eggs and early embryos was unknown, and previously identified mammalian organic osmolyte transporters could not account for their transport. Here, we report that there is a single saturable transport component shared by betaine and proline in 1-cell mouse embryos. A series of inhibitors had nearly identical effects on both betaine and proline transport by this system. In addition, K(i) values for reciprocal inhibition of betaine and proline transport were approximately 100-300 microM, similar to K(m) values ( approximately 200-300 microM) for their transport, and both had similar maximal transport rates (V(max)). The K(i) values for inhibition of betaine and proline transport by dimethylglycine were similar ( approximately 2 mM), further supporting transport of both substrates by a single transport system. Finally, betaine and proline transport each required Na(+)- and Cl(-). These data were consistent with a single, Na(+)- and Cl(-)-requiring, betaine/proline transport system in 1-cell mouse embryos. While betaine was only transported by a single saturable system, we found an additional, less conspicuous proline transport route that was betaine-insensitive, Na(+)-sensitive, and inhibited by alanine, leucine, cysteine, and methionine. Furthermore, we showed that betaine, like proline, is present in the mouse oviduct and thus could serve as a physiological substrate. Finally, accumulation of both betaine and proline increased with increasing osmolarity, consistent with a possible role as organic osmolytes in early embryos.

In utero and lactational exposure to an environmentally relevant organochlorine mixture disrupts reproductive development and function in male rats.

We hypothesized that in utero and lactational exposure of male rats to a mixture of more than 15 organochlorines, resembling that found in blubber from northern Quebec seals, alters reproductive development and function. Female rats were gavaged with either corn oil (controls) or the organochlorine mixture in increasing doses (low, medium, and high) for 5 wk before mating and through gestation. Developmental effects were monitored in the male offspring from Postnatal Day (PND) 2 until PND 90. The high-dose mixture reduced the number of pups per litter, percentage of live offspring, and pup weights (P < 0.05). Because only three rats from the high-dose treatment survived, data from this group beyond PND 2 were not included in the statistical analyses. As assessed by the time of preputial separation, puberty was delayed in the pups from treated dams (P < 0.05). Testes weights in the medium-dose group were greater than those in controls on PND 21 (P < 0.05). Ventral prostate weights were lower for the medium-dose group on PND 60 (P < 0.05). On PND 90, weights of the epididymis, ventral prostate, and seminal vesicle of the medium-dose rats were reduced compared to those of controls (P < 0.05). On PND 90, sperm motility parameters assessed by computer-assisted sperm analysis were altered in the low- and medium-dose groups (P < 0.05). Testicular and epididymal morphology was severely affected in rats exposed to the high dose of the mixture. Serum testosterone, LH, FSH, prolactin, and total thyroxine levels did not differ because of organochlorine treatment. Therefore, in utero and lactational exposure to an environmentally relevant organochlorine mixture adversely affects the reproductive system of male rats, perhaps via antiandrogenic effects during testis development, suggesting a possible reproductive health hazard for humans and other species.

Effect of mono-(2-ethylhexyl) phthalate on bovine oocyte maturation in vitro.

The effect of mono(2-ethylhexyl) phthalate (MEHP) on bovine oocyte maturation in vitro was examined. Cumulus-oocyte complexes (COCs) were cultured in maturation medium supplemented with various levels of MEHP for 24h, and then examined for the degree of cumulus expansion and the stage of maturation. A higher percentage of oocytes remained at the germinal vesicle (GV) stage after exposure to 75 and 100 micro M MEHP treatments (13.8 and 44.9% of oocytes, respectively) than the control (2.1% of oocytes). The proportion of oocytes that progressed to the metaphase II (MII) stage was significantly decreased with 25 micro M (59.6% of oocytes), 50 micro M (19.8%), 75 micro M (21.3%), and 100 micro M (3.1%) treatments than the control (77.3%). MEHP did not affect the process of cumulus expansion. For denuded oocytes, MEHP treatment of 50-100 micro M resulted in a significantly higher rate of oocytes remained at the GV stage compared to the control (53.4, 80.2, 88.4, and 5.4%, respectively). The rate of MII formation was significantly decreased with 10 micro M (60.9%) and 25 micro M (22.5%) MEHP treatments compared to control (68.9%). Furthermore, with 50, 75 or 100 micro M MEHP, no oocyte reached the MII stage. When COCs were cultured for 24h with 50 or 100 micro M MEHP and then cultured for an additional 24h in MEHP-free medium, most of the oocytes reached the MII stage (71.1 and 64.5%, respectively).Taken together, these results indicate that MEHP, at doses lower than those reported in blood transfusion patients, could negatively modulate bovine oocyte meiotic maturation in vitro, suggesting possible risks for human and other mammalians reproductive health.