1. Introduction
Early preimplantation mouse embryos are particularly susceptible to relatively small increases in external osmolarity [
1,
2,
3]. Increases in osmolarity can result in complete blocks to development in vitro, including the classic “2-cell block” in mouse embryos [
2,
4]. Because mammalian cells regulate their volumes by adjusting intracellular osmotic pressure [
5], such sensitivity to hypertonicity was linked to disruption of their ability to maintain cell volume within an acceptable range [
1,
6,
7].
The acute response of mammalian cells to a decrease in cell volume is to activate a set of transporters that act to accumulate inorganic ions, mainly Na
+, Cl
− and K
+ [
5]. Such mechanisms are active in early mouse preimplantation embryos, principally functionally coupled sodium-hydrogen and bicarbonate-chloride exchangers [
1,
8,
9,
10,
11]. However, accumulation of sufficient inorganic ions to balance external osmotic pressure can be damaging in the long term, which has led many somatic cells to employ mechanisms to accumulate benign “organic osmolytes” that can replace a portion of the inorganic ions while providing osmotic support [
12]. Characteristic organic osmolyte transporters operate in somatic cells [
13], but early embryos instead have unique organic osmolyte transporters, with the main one relying on glycine transport [
1,
3,
7,
14].
The glycine transport that is present in early embryos was attributed to “System Gly,” a classic transport activity characterized as accepting glycine and sarcosine (N-methylglycine) as high-affinity substrates and dependence on cotransport of Na
+ and Cl
− with the organic substrate [
15,
16]. System Gly activity was found to be mediated at the molecular level by the product of the
Slc6a9 gene (initially named
Glyt1) with the System Gly transporter then designated GLYT1 [
17]. Early mouse embryos have robust glycine transport that is competitively inhibited by sarcosine and is dependent on the Na
+ and Cl
− gradients [
15,
16], consistent with GLYT1 activity.
GLYT1 activity first appears during meiotic maturation of the oocyte. Germinal vesicle stage oocytes transport little or no glycine, but glycine transport reaches its maximum rate within several hours of ovulation being triggered in vivo or the oocyte being removed from the follicle in vitro [
18,
19,
20]. After GLYT1 is activated, intracellular soluble glycine levels increase from undetectable in the GV oocyte to very high levels of ~20–30 mM in second meiotic metaphase (MII) eggs and early preimplantation embryos, which is sufficient to provide substantial osmotic support [
18,
21]. The apparent GLYT1 activity is present up to about the 4-cell stage, after which it again becomes undetectable. The high intracellular glycine levels also remain through the 2-cell stage but disappear by the morula stage [
18]. Thus, GLYT1-mediated glycine transport appears to be present during the stages when embryos are most susceptible to hypertonic stress and support the accumulation of large amounts of intracellular glycine to act as an organic osmolyte. Glycine transport is present in early human embryos where it may perform a similar function [
22].
Glycine rescues the development of 1-cell mouse embryos to blastocysts in hypertonic media [
7,
14]. This ability has been attributed to GLYT1 because virtually all detectable glycine transport from the mature egg through the 4-cell stage has transport characteristics consistent with GLYT1 (above) and because the ability of glycine to rescue development at increased osmolarity was eliminated in the presence of a small molecule inhibitor that was developed to be highly selective against GLYT1 [
23]. However, it has not been shown directly that
Slc6a9 expression is necessary for glycine transport in early mouse embryos nor whether it is required for glycine to have a protective effect on embryo development under hypertonic conditions. Furthermore, whether lack of
Slc6a9 or GLYT1 activity in oocytes affects female fertility is not known. Here, we have created a conditional knockout of
Slc6a9 in oocytes to resolve these questions.
2. Materials and Methods
2.1. Chemicals and Media
Chemicals and reagents were obtained from Sigma-Aldrich (Oakville, ON, Canada) unless otherwise specified. In vitro maturation of oocytes was done in Minimal Essential Medium Alpha (MEMα; catalog #12561–056, Life Technologies, Burlington, ON, Canada) that was supplemented with cold water-soluble polyvinyl alcohol (PVA). Preimplantation embryos were cultured in modified potassium-supplemented Simplex Optimized Medium (mKSOM) with PVA instead of bovine serum albumin and omitting glutamine [
24]. The mKSOM medium contains NaCl (95 mM), KCl (2.5 mM), KH
2PO
4 (0.35 mM), MgSO
4·7H
2O (0.2 mM), Na lactate (10 mM), Glucose (0.2 mM), Na pyruvate (0.2 mM), NaHCO
3 (25 mM), CaCl
2.2H
2O) (1.7 mM), tetrasodium ethylenediaminetetraacetic acid (EDTA) (0.01 mM), K penicillin G (0.16 mM), Streptomycin SO
4 (0.03 mM), and 1mg/mL PVA. mKSOM was used at 37 °C in 5% CO
2 in air at 100% humidity. Collection of oocytes and 1-cell embryos was done using Hepes-mKSOM that was identical to mKSOM except 21 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes) was added and NaHCO
3 was reduced to 4 mM, and pH was adjusted to 7.4 at room temperature using NaOH. Components of media were embryo-tested or cell culture-tested grade. The osmolarities were 250 mOsM for mKSOM and 240 mOsM for Hepes-mKSOM (±5 mOsM). Where hypertonic mKSOM was used, its osmolarity was increased using the inert trisaccharide D(+)-raffinose as previously described [
14].
2.2. Nomenclature
Genes are identified using their standard nomenclature. Mouse lines are identified by the standard nomenclature used in the literature. The shorthand designations of each mouse line and their derivatives are used throughout (see below). For most lines, the gene and mouse line designations correspond, including Gdf9-Cre, Amhr2-Cre, and Cyp19-Cre. However, where LoxP sites were inserted in the
Slc6a9 gene, the designations use “Glyt1,” which was the original designation of this gene and still commonly used in the literature. To be consistent with previously published work [
25,
26], we have used the Glyt1 nomenclature for this line and its derivatives, while referring to the gene itself as
Slc6a9.
2.3. Animals
The University of Ottawa Animal Care Committee approved the animal usage and breeding protocols (approval numbers 3347 for breeding and 3348 for experiments), which comply with the regulations of the Canadian Council on Animal Care. Mice were maintained on a 12 h light:dark cycle with unrestricted access to water and Teklad Global 18% protein rodent diet 2018 (Envigo, Indianapolis, IN, USA).
B6D2F1 (BDF1) male mice were obtained from Charles River Canada (St. Constant, QC, Canada) and used for mating with females to produce offspring for fertility testing and 1-cell stage embryos for culture.
Glyt1tm1.2fl/fl mice (shorthand: Glyt1
fl/fl) have LoxP sites that flank
Slc6a9 gene exons 5–11 and were produced as previously described [
25,
26]. They were maintained on the C57Bl6 background.
Tg(Gdf9-icre)5092Coo/J (shorthand: Gdf9-iCre) mice express iCre driven by the mouse
Gdf9 gene promoter, restricting iCre exclusively to the oocyte [
27]. They were a gift from Dr. Barbara Vanderhyden (Ottawa, ON, Canada) and were maintained on the C57Bl6 background.
Amhr2tm3(cre)Bhr (shorthand: Amhr2-Cre) mice express Cre driven by the
Amhr2 promoter [
28].
Amhr2 is highly expressed in the granulosa cells of preantral and antral follicles as well as other reproductive tissues of Müllerian duct origin [
29,
30]. They were a gift from Dr. Barbara Vanderhyden (Ottawa, ON) and were maintained on the C57Bl6 background.
Tg(CYP19A1-cre)1Jri (shorthand: Cyp19-Cre) mice express iCre driven by the
Cyp19a1 promoter.
Cyp19a1 is highly expressed in granulosa cells of preantral and antral follicles [
31,
32]. Cyp19-Cre mice that also had the
Smarca4 (a.k.a.
Brg1) gene flanked by LoxP sites were a gift from Dr. Barbara Vanderhyden (Ottawa, ON, Canada). These were first crossed with C57Bl6/J mice to eliminate
Smarca4fl/fl and then were maintained on the C57Bl6 background.
2.4. Production of Conditional Knockouts
The Gdf9-iCre:Glyt1
fl/fl line was produced by crossing Gdf9-iCre with Glyt1
fl/fl mice. Because Gdf9-iCre is expressed in oocytes, this transgene should normally be transmitted only through males to prevent heterozygous loss of Glyt1 that occurs in offspring of GDF9-iCre:Glyt1
fl/fl females mated with wild type males [
33]. However, in the initial cycles of breeding, the Gdf9-iCre transgene was also transmitted through females to more rapidly increase the breeding stock. In this case, only Glyt1
fl/fl offspring were retained and those that were Glyt1
fl/− were not bred further (except for producing Amhr2-Cre:Glyt1
fl/− mice; see below). Generally, Gdf9-iCre:Glyt1
fl/+ males from the initial crosses were mated to Glyt1
fl/fl females to provide Gdf9-iCre:Glyt1
fl/fl males, which were paired with Glyt1
fl/fl females and maintained as breeding stock. Since the males carry one copy of the iCre transgene, the offspring were either Gdf9-iCre:Glyt1
fl/fl or Glyt1
fl/fl. Females of these genotypes were used as the experimental animals (Glyt1 knockout oocytes) and controls (Glyt1 wild type oocytes), respectively.
The Amhr2-Cre:Glyt1fl/fl line was produced by crossing Amhr2-Cre with Glyt1fl/fl mice to produce Amhr2-Cre:Glyt1fl/+ offspring. These were mated to Glyt1fl/fl to produce Amhr2-Cre:Glyt1fl/fl offspring, which were then paired with Glyt1fl/fl and maintained as breeding pairs. Because there was no oocyte expression of Cre, both males and females of the Amhr2-Cre:Glyt1fl/fl genotype could be used for breeding to produce offspring that were either Amhr2-Cre:Glyt1fl/fl or Glyt1fl/fl. Females of these genotypes were used as the experimental animals (Cre positive) and controls (Cre negative), respectively.
The Cyp19-Cre:Glyt1fl/fl line was produced using the same breeding scheme as the Amhr2-Cre:Glyt1fl/fl line. Offspring of the breeding pairs were either Cyp19-Cre:Glyt1fl/fl (experimental) or Glytfl/fl (control).
Amhr2-Cre:Glyt1fl/− mice were produced by crossing Glyt1+/− mice with Amhr2-Cre:Glyt1fl/fl mice. The Glyt1+/− mice were produced from mating Gdf9-iCre:Glyt1fl/fl females with BDF1 males. Since Gdf9-iCre:Glyt1fl/fl females have both copies of Glyt1 inactivated in their oocytes, they produce offspring with one copy of Glyt1 inactivated (Glyt1+/−) when mated with wild type males. Those Glyt1+/− offspring that did not inherit Gdf9-Cre were selected and mated with Amhr2-Cre:Glyt1fl/fl mice to produce offspring with Amhr2-Cre:Glyt1fl/+ and Amhr2-Cre:Glyt1fl/− genotypes as well as the corresponding Amhr2-Cre negative genotypes (Glyt1fl/+ and Glyt1fl/−).
2.5. Genotyping
Genotyping was done using ear notch samples. PCR products were visualized on 1.4% agarose gels with ethidium bromide. Band sizes were estimated from DNA ladders (ThermoFisher Scientific #SM0403). The lower set of resolved marker bands range from 100 bp to 900 bp at 100 bp intervals and then 1031 bp; the upper set was 1500 bp, 2000 bp, and 2500 bp, then 3000 bp to 10,000 bp at 1000 bp intervals, the upper range of which was generally not fully resolved. Glyt1fl/fl genotyping of offspring was done using the 17854 sense primer (5′-TGG CAC CTC TCT GAG TGT GC-3′) and 17672 antisense primer (5′- TTC CAG GAC ATC CAG ATG ATG C - 3′) that yield bands of 258 bp for Glyt1fl/fl and 182 bp for wild type.
Determinations of whether Cre-mediated deletion of a segment of
Slc6a9 had occurred in Glyt1
fl/fl tissues was done using the o250 sense primer (5′-CCC ATG CCC AGA TCC ATG C-3′) targeted 5′ to the LoxP site and the o228 antisense primer targeting
Slc6a9 sequence 3′ to the PGK-neomycin cassette (5′-GTC AAC CTG ACT CCT AGC CCT GTA CC-3′) as previously described [
25]. In the absence of Cre, this yields a product of ~6600 bp. With successful Cre-mediated deletion, a band of ~450 bp is produced instead. This was performed on ovary and liver samples to assess the effects of Cre targeted to granulosa cells.
The Gdf9-iCre transgene was detected using the primer sets specified by The Jackson Laboratory (
https://www.jax.org/Protocol?stockNumber=011062&protocolID=17814 (accessed on 4 September 2023)). The iCre transgene was detected with forward primer 25494 (5′-GGC ATG CTT GAG GTC TGA TTA C-3′) and reverse primer 21218 (5′-CAG GTT TTG GTG CAC AGT CA-3′), which were multiplexed with the Internal Positive Control (targeting the mouse
Il2 gene) forward primer oIMR7338 (5′-CTA GGC CAC AGA ATT GAA AGA TCT-3′) and reverse primer oIMR7339 (5′-GTA GGT GGA AAT TCT AGC ATC ATC C-3′). The Cre transgene primers yield a 200 bp product and the positive control primers yield 324 bp.
The Amhr2-Cre transgene was detected with the forward primer AMHR2Cre FWD (5′-CTC TGG TGT AGC TGA TGA TC-3′) and reverse primer AMHR2Cre REV (5′-TAA TCG CCA TCT TCC AGC AG -3′), which were multiplexed with the Internal Positive Control primer IMR0015 (5′-CAA ATG TTG CTT GTC TGG TG-3′) and reverse primer IMR0016 (5′-GTC AGT CGA GTG CAC AGT TT-3′). The Cre transgene was indicated by a 340 bp product and the positive control product was 206 bp.
The Cyp19-Cre transgene was detected with the forward primer Cyp19CreF (5′-ACTTGGTCAAAGTCAGTGCG-3′) and reverse primer Cyp19CreR (5′-CCTGGTGCAAGCTGAACAAC-3′), which yields a 290 bp product. No internal control was used for Cyp19-Cre, so these primers were always paired with the Glyt1fl/fl primers (above) to ensure DNA quality.
2.6. GV Oocytes and 1-Cell Stage Embryos
To obtain germinal vesicle stage (GV) oocytes, females were superovulated with 5 IU equine chorionic gonadotropin (eCG, Prospec, Sturgeon County, AB, Canada) administered by intraperitoneal (IP) injection. Ovaries were excised and minced in Hepes-KSOM at 44–46 h post-eCG. Cumulus-oocyte complexes (COCs) were collected, and cumulus cells were removed by repeated pipetting to obtain denuded oocytes.
One-cell stage embryos were obtained by inducing ovulation with an IP injection of 5 IU human chorionic gonadotropin (hCG, Prospec) at 47 h post-eCG. Females were then caged overnight with BDF1 males (Charles River Canada) following hCG injection. Embryos were obtained 21–24 h post-hCG by flushing oviducts with Hepes-KSOM medium using a blunt-end syringe.
2.7. Glycine Transport Measurements
The methods for measuring GLYT1 activity have been extensively described and validated previously [
18,
19,
20,
23,
34]. [
3H]glycine ([2-
3H]glycine; 40–60 Ci/mmole) was obtained from Perkin-Elmer (Waltham, MA) and used where indicated. During the course of this work, [
3H]glycine that passed quality control tests (see below) became unavailable from this source. After that, [
3H]glycine ([2-
3H]glycine; 2.4 Ci/mmole) was obtained from Movarek (Brea, CA) and used where indicated. Each new stock of [
3H]-glycine was subject to a quality control test that confirmed that transport of 1 µM [
3H]-glycine into mouse 1-cell embryos was inhibited by >90% in the presence of 5 mM unlabelled glycine.
Glycine transport into individual denuded oocytes was measured by incubating oocytes in 3 µM [3H]glycine (Perkin-Elmer) after they had been in vitro matured overnight in MEMα medium to MII eggs to allow GLYT1 activity to reach maximum levels. Glycine transport into COCs was measured using 1 µM [3H]glycine from Perkin-Elmer or 10 µM of the lower specific activity [3H]glycine from Movarek. It was confirmed using remaining Perkin-Elmer stock that had passed the quality control test that essentially the same results were obtained when using [3H]glycine from either source. Glycine transport measurements were carried out in 50 µL drops of mKSOM under mineral oil with a 10 min incubation with [3H]glycine.
After incubation with [3H]glycine, denuded oocytes or COCs were washed 7× in ice-cold Hepes-mKSOM and placed into scintillation vials with 4 mL Scintiverse BD scintillation fluid (Fisher Scientific, Pittsburgh, PA). An LS6500 liquid scintillation counter (Beckman Coulter, Brea, CA) set at a 5 min counting period was used to quantify the accumulated [3H]glycine. CPM were converted to molar amounts of [3H]glycine using standard curves constructed from serial dilutions of [3H]glycine stocks. Backgrounds with no added [3H]glycine were subtracted from each reading. Rates of glycine transport were reported as fmole [3H]-glycine per oocyte per min per µM [3H]-glycine in the incubation medium.
Transfer of [3H]glycine from cumulus cells into the enclosed oocyte was assessed by incubating both COCs and denuded GV oocytes for 4 h in mKSOM with 10 µM [3H]glycine (Movarek). After the incubation, cumulus cells were removed from the oocyte in COCs by repeated pipetting though a narrow bore pipette. The amount of [3H]glycine that had been accumulated in oocytes that were within COCs vs. those that were denuded oocytes during the 4 h incubation was then determined by liquid scintillation counting as described above.
2.8. Data Analysis
Data were analyzed and graphed using GraphPad Prism 9 or 10 (San Diego, CA, USA). The statistical significance of differences between means was determined by one-way ANOVA with Tukey’s multiple comparisons test for more than two groups. A t-test was used for two groups (or a Mann-Whitney test for non-normally distributed data). One-sample t-tests were used to test for significant difference of means from zero. Statistical significance was taken to be p < 0.05.
4. Discussion
Glycine is the major organic osmolyte in preimplantation mouse embryos, where it plays a key role in maintaining cell volume [
7,
15,
23,
36]. It likely has a similar role in the embryos of other mammals including humans [
22]. In the absence of organic osmolytes supplied from the external environment, relatively minor increases in osmolarity can result in developmental arrest in early embryogenesis [
1,
2,
4,
14,
16]. It had been established that glycine is transported into mouse embryos during the 1-cell to 4-cell stages by a transporter that had the characteristics of the classic System Gly that is mediated by the GLYT1 transporter encoded by
Slc6a9. The evidence for this included competitive inhibition by sarcosine, dependence on both Na
+ and Cl
˗, and inhibition by a small molecule inhibitor, ORG23798, that is selective for GLYT1 [
15,
16,
23]. However, it had not been shown that a functional
Slc6a9 gene is required for glycine transport into early preimplantation embryos. Here, we were able to create an oocyte-specific knockout of
Slc6a9 by crossing mice in which a segment of the gene had been flanked by LoxP sites [
25,
26] with transgenic mice expressing iCRE driven by the
Gdf9 promoter [
27]. Offspring of Gdf9-iCre:Glyt1
fl/fl females mated to wild type males were all heterozygous for
Slc6a9, indicating that all oocytes that gave rise to these offspring were
Slc6a9−/−. Furthermore, when matured, such oocytes all failed to transport glycine, confirming that
Slc6a9 in necessary for the robust glycine transport found in eggs and persisting in early preimplantation embryos.
Unexpectedly, female Gdf9-Cre:Glyt1
fl/fl females were fertile, producing litters whose sizes and frequency were indistinguishable from those of Glyt1
fl/fl females. There are several possible explanations for this result. The first is that glycine is not required by oocytes and early preimplantation embryos in vivo. There is considerable evidence that glycine is used by mouse embryos as an organic osmolyte and rescues development from the developmental block that occurs when osmolarity is increased in vitro [
2,
7,
14,
15,
23,
36]. However, it had not been shown that glycine is similarly required for preimplantation development in vivo. It is possible that it is used by embryos only under conditions of osmotic stress (e.g., dehydration), but this would not explain why embryos contain such high levels of endogenous glycine [
18] when mice were housed under normal laboratory conditions with ad libitum access to water.
A second possibility is that glycine is supplied to embryos through another route that compensates for the loss of GLYT1 activity in the embryos themselves. We showed here that glycine taken up by cumulus cells can supply glycine to the enclosed oocyte within a COC, which supports the hypothesis that cumulus-mediated glycine transport can compensate at least partly for the loss of GLYT1 in the egg and early preimplantation embryo. The large majority of glycine transport by the cumulus is also via GLYT1 [
35]. However, attempts to eliminate GLYT1 activity in cumulus cells by disrupting the
Slc6a9 gene in granulosa cells using Cre expression driven by the
Amhr2 promoter in either homozygous Glyt1
fl/fl females or heterozygous Glyt1
fl/− females lacking one copy of the
Slc6a9 gene were unsuccessful, as were attempts using Cre expression driven by the
Cyp19a1 promoter. Therefore, it could not be determined whether eliminating both routes of glycine accumulation via GLYT1 would affect female fertility. However, the accumulation of glycine in
Slc6a9−/− oocytes within COCs supports the hypothesis that GLYT1 in cumulus cells compensates for its loss in eggs and early embryos and that it may contribute to maintaining female fertility.
A third possibility is that organic osmolytes other than glycine contribute to volume regulation of eggs and early embryos. In addition to glycine, betaine (N,N,N-trimethylglycine) is a major osmolyte in mouse eggs and preimplantation embryos. It is synthesized in oocytes during meiotic maturation by the enzyme choline dehydrogenase (CHDH) where it is accumulated to very high levels in the mature egg [
37]. Its intracellular concentration is then regulated in 1-cell and 2-cell embryos by the SIT1 (
Slc6a20a) transporter [
38,
39]. Thus, increased betaine synthesis or transport could compensate for decreased glycine in embryos. Whether knocking out the betaine pathways in conjunction with knocking out GLYT1 activity affects female fertility remains to be determined, however.
Another consideration is that different strains of mice have very different sensitivities to increased osmolarity. Mice of different strains exhibit widely varying susceptibility to the classic “2-cell block” to development, ranging from those that suffer a near-total block in traditional embryo culture media to classic “non-blocking” strains that developed to blastocysts in vitro at high rates [
40,
41]. We had previously shown that the block that occurred when osmolarity was increased was identical to the 2-cell block, with some strains becoming blocked at near-oviductal osmolarities, while others only became blocked at much higher osmolarities [
2,
4]. The C57Bl6 mice used in the current investigations become blocked at intermediate osmolarities beginning around 330 mOsM, between sensitive strains that become blocked at <310 mOsM and resistant strains that only become blocked at >350 mOsM. It is possible, therefore, that the fertility of more sensitive strains might be affected by lack of GLYT1 activity in oocytes. This remains to be investigated.
A requirement for GLYT1 activity in preimplantation embryos for glycine to mitigate against the deleterious effects of increased osmolarity was confirmed. As was previously shown, 1-cell stage embryos would not develop at increased osmolarity, but development could be rescued with glycine added to the medium [
2,
23]. The ability of glycine to rescue development was lost, however, in embryos in which
Slc6a9 was disrupted, conclusively demonstrating the requirement for GLYT1. Although we used 370 mOsM medium here to obtain a maximum difference between development with and without glycine, a significant decrease in development was evident between 310 and 330 mOsM. This is nearer to the in vivo osmolarity of oviductal fluid, which has been measured to be ~300 mOsM [
42,
43,
44], although calculations have indicated it may reach as high as 350 mOsM during early preimplantation embryo development [
3]. This is supportive of a possible role for glycine in vivo, since relatively small increases in oviductal fluid osmolarity would have an impact on embryo development even at the lower values proposed for oviductal fluid. A role for glycine in vivo is also implied by the large intracellular concentration of glycine in the egg through 2-cell embryo stages [
18,
21], which can be maintained against a steep outward gradient only at considerable and continuous metabolic cost.
Ample evidence suggests that organic osmolytes such as glycine are beneficial to early preimplantation embryos in vitro [
1]. This likely includes human embryos, since they transport glycine via GLYT1 similarly to mouse embryos [
22]. Current embryo culture media in use in the clinic include glycine and other amino acids and, therefore, support cell volume regulation that requires organic osmolytes. The results presented here indicate the need for glycine in media used for in vitro oocyte maturation as well, to support the accumulation of glycine in the mature egg.
In summary, we have shown conclusively that a functional Slc6a9 gene is required for GLYT1 activity in mouse oocytes. Additionally, it is required for the rescue by glycine of development at increased osmolarities in vitro. At least in the C57Bl6 strain, GLYT1 in oocytes and preimplantation embryos is not required for female fertility. We propose that this is likely due to redundant mechanisms that include glycine transport into oocytes via cumulus cells and compensation by other organic osmolytes such as betaine. Future studies will be needed to develop a method of also knocking out glycine transport in cumulus cells, to determine whether this would decrease fertility. Also, it remains to be determined whether preventing the use of other organic osmolytes such as betaine in oocytes in conjunction with knocking out GLYT1 activity results in impaired fertility.